[48]
AMINOGI.,YCOSIDE-MODIFYING ENZYMES
611
[48] Aminoglycoside-Modifying Enzymes B y MICHAEL J. HAAS and JOHN E. DOWDING
This article describes the isolation and assay procedures currently used in the study of the nine aminoglycoside-modifying activities listed in the table. It should be emphasized that very little information is available for some of the enzymes listed in the table and for this reason independently isolated activities catalyzing the same reaction have been grouped together (e.g., the three kanamycin acetyltransferases) pending studies showing them to be substantially different enzymes. These enzymes, which modify the aminoglycoside or aminocyclitol antibiotics, have been detected in a wide variety of resistant bacteria. In many clinical isolates they are known to be plasmid-coded, and in certain strains the enzymes appear to be located near the cell surface) a fact which is used to advantage in some of the enzyme isolation techniques described below. Among Eubacteria the three known aminoglycoside-modification mechanisms are acetylation of amino groups and phosphorylation and adenylylation of hydroxyl groups; these mechanisms have recently been reviewed by Benveniste and Davies. 1 Other modified compounds have been detected among antibiotic-producing organisms,2, 3 and it is possible that in these strains these may be biosynthetic intermediates or inactivation products. The structures of the aminoglycoside antibiotics (with positions of modification) are shown in Figs. 1-7. Although an indication of the modification site may often be obtained by examination of the substrate specificity of an enzyme, it has been determined in most cases by chemical and/or spectroscopic analysis of the modified antibiotic (table). The study of these enzymes is particularly valuable for two reasons. First, determinations of the type and site of modification have allowed the design and synthesis of semisynthetic antibiotics not modified by the enzyme and therefore active against many resistant isolates. Second, the enzymes provide a very sensitive, rapid and often specific assay for the aminoglycoside antibiotics2, ~ It is important to remember that many R ]actor-containing strains
1R. Benveniste and J. Davies, Annu. Rev. Biochem. 42, 471 (1973). "~M. K. Majumdar and S. K. Majumdar, Biochem. J. 120, 271 (1970). M. Kojuma, S. Inouye, and T. Niida, J. Antibiot. 26, 246 (1973). 4j. Davies, M. Brzezinska, and R. Benveniste, Ann. N.Y. Acad. Sci. 182, 226 (1971), 5M. J. Haas and J. Davies, Antimicrob. Ag. Chemother. 4, 497 (1973).
612
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
/b HO,--x--.~C,H,~O\
o"
I- b
d
7
~
C H o ~ N H R
3
0
~o~H~ H O ~
Kanamycin A Kanamycin B Kanamycin C BB-K8 (amikacin)
Rl
R~
NH~ NH~ OH NH~
OH NH2 NH: OH
OH OH R3
H H H --CO---CH(OH)CH:--CH2NH~
Fro. 1. Structure of the kanamycins. Arrows indicate sites of O-phosphorylation (a), N-acetylation (b, c, d), and O-adenylylation (e).
are pathogens or potential pathogens. Care should be exercised when handling cultures and all used media and glassware should be sterilized be]ore disposal or cleaning.
Assay Procedures General Protocol. Enzyme activities are assayed by means of the phosphocellulose paper binding assay devised by Davies et al. 4 which measures transfer of radiolabel from a suitable cofactor to the antibiotic. The general assay procedure used is outlined below and is followed by details for each type of reaction. The reaction mixture consists of buffer, labeled cofactor, enzyme prepa s
I
0 NH,7
~
NH~
O OH
t
d
Fxo. 2. Structure of tobramycin (nebramycin factor 6). Arrows indicate sites of N-acetylation (a, b, c) and O-adenylylation (e).
[48]
AMINOGLYCOSIDE-MODI FYI NG ENZYMES
.R, -
613
/b
S(~H-R~,-'I~O \
d
o R~
/
e
Gentamicin Gentamicin Gentamicin Gentamicin Gentamicin
A B CI~ C~ C1
R1
R~
R3
R4
R5
R6
R7
H H H CH3 CHa
OH NH2 NH~ NH~. NHCHa
OH H H It H
OH OH H H H
NH2 OH NH2 NH~ NH..
H OH OH OH OH
OH CHa CHa CH3 CH3
FxG. 3. Structure of the gentamicins. Sisomicin is 4,5-dehydrogentamicin C1, (ring I). Arrows indicate sites of O-phosphorylation (a), N-acetylation (b, c, d), and O-adenylylation (e). a r a t i o n a n d a n t i b i o t i c . A c r u d e e n z y m e e x t r a c t (S100 or o s m o t i c shocka t e ) is of s u i t a b l e p u r i t y for t h e a s s a y . F o r large n u m b e r s of a s s a y s it is c o n v e n i e n t to d i s t r i b u t e p r e m i x e d buffer, c o f a c t o r , a n d e n z y m e (or buffer, cofaetor, a n d a n t i b i o t i c ) . Such m i x t u r e s s h o u l d n o t be r e - u s e d b
HO
6CHL'N/H O.
R3
Ribostamycin Butirosin A Butirosin B
OH
R,
1{2
R3
H - - C O--C H (O H) - - C H r--C H..,NH.., --CO--CH(OH)--CH2--CH~NH2
H OH H
OH H OH
FIG. 4. Structures of ribostamycin and the butirosins. Arrows indicate sites of O-phosphorylation (a) and N-acetylation (b, c, d).
614
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
/b 0 ,-.~,^
c
o"7" ~ - - - ~ --.~'. %
I).o\?-..y6.
'~ '2
R,~
Rl
R~
Neomycin B Neomycin C
NH2 NH2
OH OH
Paromomycin
OH
OH
Lividomycin A Lividomycin B
OH OH
H H
0
---~-
=
OH
R8
R4
H CH2NH2 H + {CH.NH~ H H
R~
CH~NH2 H
H H
CH~NH2}
H
CH2NH2 CH:NH2
mannose H
FIe. 5. Structures of the neomycins and lividomycins. Neamine (and paromamine) are rings I and II. Arrows indicate sites of O-phosphorylation (a) and N-acetylation (b, c, d). NH II HNCNH2
NH CNH2
0
H
R~
OH
Ii"
OH ~) R=O C~H~HO4 /OH
Streptomycin Dihydrostreptomycin Mannosidostreptomycin
RI
R2
CHO CH2OH C HO
H H mannose
Fro. 6. Structure of the streptomycins. Ring I is streptidine, ring II is streptose, and ring III is N-methyl4.-glucosamine. The arrow indicates the site of O-adenylylation or phosphorylation.
[481
AMINOGLYCOSIDE-MODIFYIN G ENZYMES
~
()l;~
CH3
0 FIG. 7. Structure of speetinomycin. The arrow indicates the site of O-adenylylation. after refrigeration or freezing. Control (blank) assays lacking either antibiotic or enzyme should be included in each set of assays. The components of the assay are mixed in chilled 75 X 10 mm tubes and the reaction is started by incubation at 30 ° or 35 °. After 15-20 rain of incubation the tubes are returned to the ice bath and a 10-20 ~1 sample of each reaction mixture is pipetted onto a numbered piece (ca. 1 cm ~) of W h a t m a n P-81 phosphocellulose paper raised above a block of styrofoam on a pin. After 30 sec at room temperature papers and pins are placed in about 500 ml of hot (70--80 °) distilled water for 3-4 min. The liquid, which contains unused labeled cofactor, is poured off and disposed of accordingly. The papers are rinsed 3 or 4 times with 500-600 ml of distilled water and dried under heat lamps (avoid charring) or using a hot-air dryer. Papers (with or without pins) are placed in glass scintillation vials containing 10 ml of toluene scintillation fluid (3 g PP(), 0.1 g P O P O P per liter of toluene) and counted. A time course of inactivation may be determined by simply scaling up the reaction mixture and removing samples for counting at various times. Descriptions of large-scale inactivations for chemical analyses m,~y be found among the references in the table. Assay o.f Kanamycin and Gentamicin Acetyltrans]erases. Acetyltransferases with acid pH optima are assayed in a buffer prepared by adding to 30 ml of deionized water: 12.75 ml of 0.6 M citric acid, 27.25 rnl of 0.6 M tripotassium citrate, 2.4 ml of 1.0 M magnesium acetate, and 1.6 ml of 0.5 M D T T . The pH is adjusted to the desired value at 30 ° with potassium hydroxide or glacial acetic acid and the volume is adjusted to 80 ml with deionized water. Acetyltransferases with neutral or slightly basic pH optima are assayed in a buffer prepared by adding to 40 ml of deionized water: 25 ml of 1 M Tris base, 5 ml of 1 M MgCl~, 1 ml of 0.5 M D T T and 10 ml of 1 M NH4C1. The pH is adjusted to the desired value at 30 ° with HC1 or glacial acetic acid and the volume is adjusted to 100 ml with deionized water.
616
ANTIBIOTIC INACTIVATION AND MODIFICATION A M I N O G L Y C O S I D E - M ODIFYING
Enzyme-
[48]
ENZYMES
Synonyms
Substrates b
Kanamycin acetyltransferase (KAT)
--
Gentamicin acetyltransferase I (GATI)
--
Neomycins, kanamycins A and B, gentamicin Cla, tobramycin, butirosins, ribostamycin, sisomicin, BB-K8 (gentamicin C2) Gentamicins, sisomicin (kanamycin B, tobramycin)
Gentamicin acetyltransferase II (GATH)
Gentamiein acetyltransferase I I I (GATm) Gentamicin adenylyltransferase (GAdT) Streptomycin-spectinomycin adenylyltransferase (SAdT) Neomycin phosphotransferase I (NPTI) Neomycin phosphotransferase II (NPT~) Streptomycin phosphotransferase (SPT)
Gentamicin adenylate synthetase (GAS), kanamyein nucleotidyltransferase (KNT) k Streptomycin adenylate synthetase (SAS) Kanamyain phosphotransferase I (KPTI) n, possibly also lividomycin phosphotransferase (LPT) Kanamycin phosphotransferase II (KPTII) n
Kanamycin C, gentamicins, sisomicin, tobramycin, butirosins (neomycins, ribostamycin kanamycin B) Kanamycins, gentamicins, sisomicin, ribostamycin, tobramycin, lividomycins Kanamycins, gentamicins, tobramycin Streptomycin, spectinomycin, dihydrostreptomycin (mannosidostreptomycin) Neomycins, kanamycins, lividomycins, ribostamycin, gentamicins A and B Neomycins, kanamycins, butirosins, ribostamycin, gentamicins A and 'B Streptomycin, dihydrostreptomycin
"Enzyme names and abbreviations are currently under review. b Poor substrates are shown in parentheses. c Aminoglycoside structures are shown in Figs. 1-7. d H. Umezawa, M. Okanishi, R. Utahara, K. Maeda, and S. Kondo, J. Antibiot. 20, 136 (1967). M. Yagisawa, H. Naganawa, S. Kondo, T. Takeuchi, and H. Umezawa, J. Antibiot. 25, 495 (1972). I R. Benveniste and J. Davies, Proc. Nat. Acad. Sci. U.S. 70, 2276 (1973). g M. Brzezinska, R. Benveniste, J. Davies, P. J. L. I)aniels, and J. Weinstein, Biochemistry 11, 761 (1972). h M. Chevereau, P. J. L. Daniels, J. Davies, and F. Le Goffic, Biochemistry 13, 598 (1974). i S. Biddlecome, P. Daniels, J. Davies, M. Haas, and D. Rane, in preparation. i H. Naganawa, M. Yagisawa, S. Kondo, T. Takeuchi, and H. Umezawa, J. Antibiot. 24, 913 (1971).
[48l
AMINOGLYCOSIDE-MODIFYING ENZYMES
Cofactors
Modificationc
617
Representative strains ~
Acetyl coenzyme A
6-Amino group of aminohexose I is aeetylated a,e,/
Acetyt coenzyme A
3-Amino group of deoxystreptamine (II) is acetylatedg
Acetyl coenzyme A
2-Amino group of aminohexose I is acetylated/,h
Acetyl coenzyme A
3-Amino group of deoxystreptamine (II) is acetylated ~
P. aeruginosa PST 1*
Ribo- or deoxyribonucleoside triphosphates (ATP preferred)
2-Hydroxyl group of aminohexose I I I is adenylylatedi
E. coli JR66/W677
ATP or dATP m
3-Hydroxyl group of Nmethyl-I~glucosamine is adenylylated z 3-Hydroxyl group of aminohexose I ° or 5-hydroxyl group of pentose I I I ~ is phosphorylated 3-Hydroxyl group of aminohexose I is phosphorylated
E. coli RlOO/W4354," NR73/W677
ATP
ATP ATP
3-Hydroxyl group of Nmethyl-L-glucosamine is phosphorylated~
E. coli R5/W677~; NR79/ W677, P. aeruginosa GN315/3796, Streptomyces kanamyceticus ATCC 12853/ P. aeruginosa 1 3 0 / P . aeruginosa 2 0 9 / E . coli R135/C600 Providencia 164,h Streptomyces spectabilis UC 2472/
E. coli JR35/W677p
E. coli JR66/W677,q P. aeruginosa Ps49q E. coli J R 3 5 / W 6 7 7 / P. aeruginosa H-9, r Staphylococcus aureus B294," Streptomyces griseus ATCC 10971t
k S. Kondo, K. Iinuma, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 27, 90 (1974). z T. Yamada, D. Tipper, and J. Davies, Nature (London) 219, 288 (1968). " R. Benveniste, T. Yamada, and J. Davies, Infection Immunity 1, 109 (1970). " H. Umezawa, H. Yamamoto, M. Yagisawa, S. Kondo, T. Takeuchi, and Y. A. Chabbert, J. Antibiot. 26, 407 (1973). ° S. Kondo, M. Okanishi, R. Utahara, K. Maeda, and H. Umezawa, J. Antibiot. 21, 22 (1968). p B. Ozanne, R. Benveniste, D. Tipper, and J. Davies, J. Bacteriol. 100, 1144 (1969). q M. Brzezinska and J. Davies, Antimicrob. Ag. Chemother. 3, 266 (1973). r O. Doi, M. Ogura, N. Tanaka, and H. Umezawa, Appl. Microbiol. 16, 1276 (1968). O. Doi, M. Miyamoto, N. Tanaka, and H. Umezawa, Appl. Microbiol. 16, 1282 (1968). t j. Walker and M. Skorvaga, J. Biol. Chem. 248, 2435 (1973). " Cultures not available from culture collections may usually be obtained from authors of relevant publications.
618
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
Lyophilized [1-14C]acetyl coenzyme A (50-60 Ci/mole) is resuspended in deionized water to an activity of 25 ~Ci/ml and diluted with 2 parts of 0.88 mg/ml trilithium acetyl coenzyme A before use. Each assay contains: 10 t~l of buffer, 10 t~l of [14C]acetyl-CoA solution, 5 ~l of enzyme preparation, 5 ~l of antibiotic (1 mg/ml). The mixture is incubated at 30 ° for 15 min and 20-~1 samples are counted. If diluted or less active enzyme preparations are being assayed, up to 20 t~l may be used in each assay. Assay o] Neomycin and Streptomycin Phosphotrans]erases. To prepare 30 ml of buffer (sufficient for 3000 assays), mix 15 ml of distilled water with 2 ml of 1 M Tris, 1.25 ml of 1 M MgCl~, 6 ml of 2 M NH4CI, and 0.1 ml of 0.5 M DTT. Adjust the pH to 7.1 at 5 ° with 1 M maleic acid and make up to 30 ml with deionized water. [~2P]ATP solution consists of 15 ~l of 50 mM sodium ATP (adjusted to pH 7.2 with NaOH) and 10-100 ~1 of [~/-~P]ATP depending on specific activity (initially-~ 20 Ci/mole, 0.75 mCi/ml) made up to 1 ml with deionized water. Each assay contains: 10 ~l of buffer, 10 ~l of [3-°P]ATP solution, 10 t~l of enzyme preparation, 2 ~l of antibiotic (1 mg/ml). The mixture is incubated at 35 ° for 20 min, and 20-~1 samples are counted. COMMENTS. Owing to the short half-life of 32p, ATP solutions should be used within 2-3 weeks of preparation. Assays o/ Gentamicin and Streptomycin Adenylyltrans]erases. The assay buffer is the same as for phosphotransferase assays except that it is brought to pH 7.8 at 5 °. Uniformly labeled [~C]ATP (400-500 Ci/mole) is purchased in 50% ethanol, lyophilized to dryness and resuspended in deionized water to an activity of 50 ~Ci/ml. To 1 ml of this are added 50 ~l 0.1 M ATP and 3.95 ml of deionized water, yielding a final cofactor preparation of approximately 10 t~Ci/tLmole/ml. Each assay contains: 10 ~l of buffer, 10 ~l of [~4C]ATP solution, 10 ~l of enzyme preparation, 2 ~l of antibiotic (1 mg/ml). The mixture is incubated at 30 ° for 30 min and 20- or 25-~1 samples are counted. COMMENTS. [14C]ATP may be purchased in 2% ethanol, when it is used directly after dilution. [a-32P]ATP may also be used as an adenyl donor but the short half-life of 32p is usually a disadvantage. A number of other methods have been used to assay inactivation by aminoglycoside-modifying enzymes, the most common being a microbiological assay which measures residual antibiotic activity by disc assay against a sensitive test organism2 Although this assay is convenient and T. Gavan, E. Cheatle, H. McFadden, Jr., Eds., "Antimicrobial Susceptibility Testing." American Society of Clinical Pathologists, Chicago, Illinois (1971).
[481
A M I N O G L Y C O S I D EODIFYI -M NG ENZYMES
619
simple, it may not be sufficiently quantitative for some experiments (e.g., time courses of inactivation). In addition, it cannot measure modification of a compound that is not an antibiotic. Furthermore, modification may not be detected if it does not result in complete inactivation (e.g., 6'-Nacetylneomycin B retains considerable antimicrobial activity). N-Acetylation may be monitored by means of a colorimetric assay using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). 7 Free eoenzyme A formed during acetylation reacts with DTNB to form a mixed disulfide; this is detected by an increase in A41_~. The assay is useful for certain antibiotics which do not bind quantitatively to phosphocellulose paper or if radiolabeled acetyl CoA is unavailable, but it requires the use of at least partially purified enzyme preparations. Sulfhydryl reagents interfere with the DTNB assay. The products of modification reactions may also be identified by cellulose-acetate strip electrophoresis 7 and thin-layer chromatography ;~ residual antibiotic can be determined by high-pressure liquid chromatography, s Growth of Cells
Drug-resistant bacteria are grown at 37 ° in medium containing, per liter: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride. Ten grams of glucose are added separately, as a sterile 50% solution, after sterilization of the medium. One of the antibiotics to which the R factor confers resistance is routinely added to the medium after sterilization (to a final concentration of 20 ~g/ml) to prevent possible segregation of the R factor. Streptomycetes are grown for 18-36 hr at 30 ° in Difco Nutrient Broth supplemented after sterilization with glucose (final concentration 1%).
Buf]ers Buffer I: 10 mM Tris-chloride, 10 mM magnesium chloride, 25 mM ammonium chloride, 0.6 mM fl-mercaptoethanol, pH 7.8 at 4 ° Buffer II: 20 mM Tris-chloride, 10 mM magnesium acetate, 25 mM ammonium chloride, 10 mM potassium chloride, 2 mM DTT, pH 7.5 at 4 ° Buffer III: 10 mM Tris-acetate, 10 mM magnesium acetate, 1 mM fl-mercaptoethanol, pH 7.4, at 4 °. pH is adjusted with acetic acid. Buffer IV: 20 mM Tris-chloride, 10 mM magnesium chloride, 10 mM potassium chloride, 30% glycerol, pH 7.8 R. Benveniste and J. Davies, Biochemistry 10, 1787 (1971). s H. Umezawa, H. Yamamoto, M. Yagisawa, S. Kondo, T. Takeuchi, and Y. Chabbert, J. Antibiot. 26, 407 (1973).
620
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
Buffer V: 10 mM Tris-chloride, l0 mM magnesium chloride, 3 mM, fl-mercaptoethanol, pH 7.8
Isolation of Crude Enzyme Preparations For those species (Escherichia, Shigella, Enterobacter, Serratia, Salmonella, and some strains of Pseudomonas) which release their drugmodifying activity when subjected to an osmotic shock, the procedure of Neu and Heppel 9 is used to obtain crude enzyme preparations. This technique solubilizes essentially all the aminoglycoside activity of an R ÷ E. coli while releasing only 4-6% of the total cell protein. One to two percent of the cells are lysed duriag such treatment. Where purification of an enzyme is desired, it is obviously an advantage if the R factor can be transferred to a strain susceptible to osmotic shock. 1° Aminoglycoside-modifying enzymes may be isolated from strains not susceptible to osmotic shock (notably Proteus, Providencia, Staphylococcus, Streptomyces, and some strains of Pseudomonas) either by sonication, French pressing, or enzymic lysis of cells. Osmotic Shock. Late log phase cells are harvested by centrifugation and resuspended in 100 volumes of 10 mM Tris-chloride, 30 mM sodium chloride, pH 7.8 at room temperature. The cells are again harvested by centrifugation, resuspended in 35 volumes of 3 mM EDTA, 33 mM Trischloride, pH 7.8, in 20% sucrose and stirred for 15 min at room temperature (a magnetic stirring bar is convenient). The cells are centrifuged at 16,000 g for 15 min in a refrigerated centrifuge, and the supernatant is removed. The centrifuge tube is inverted, and the pellet is allowed to drain in the cold for 10 min. It is important to remove all traces of sucrose or the cells will not shock properly. Therefore all excess fluid is removed from the cell pellet with cotton or other absorbent material. The ceils are resuspended in 40 volumes of ice-cold 0.5 mM m~gnesium chloride, stirred at 4 ° for 10-20 min and then centrifuged at 26,000 g for 30 min at 4 °. The pellet is discarded; the supernatant ("osmotic shockate") contains the aminoglycoside-modifying enzymes. Sonication. Washed log phase cells are resuspended in an appropriate buffer (0.6 g wet cells to 8 ml of buffer). The suspension, cooled in an ice bath, is subjected to 30-see bursts from a sonicator at 100 W output. Bursts are alternated with 2-min cooling periods, and 3-5 bursts are usually adequate (cell breakage may be monitored by phase-contrast microscopy). Some staphylococci cannot be broken by sonication; others may reH. C. Neu and L. A. Heppel, J. Biol. Chem. 240, 3685 (1965). 1, j. Davies, this volume [3].
[4sl
AMINOGLYCOSIDE-MODIFYING ENZYMES
621
quire the use of glass powder and longer sonication times for efficient breakage. Streptomycetes are resuspended in a minimum volume of Buffer I and sonicated as above. Sonicated cell suspensions are centrifuged at 30,000 g for 20-30 min, and the supernatant is dialyzed. The extract may be further clarified by preparing a 100,000 g supernatant (S100); deoxyribonuclease I is added to the 30,000 g supernatant to a final concentration of 4 ~g/ml, and the mixture is centrifuged at 100,000 g for 2 hr at 4 °. The supernatant is dialyzed against an appropriate buffer (usually buffer I or buffer II--see above). French Pressure Cell. The French pressure cell is the most convenient method of breaking large quantities of cells. The cells from 6 liters of a log phase culture are harvested by centrifugation, resuspended in 1 liter of 10 mM phosphate buffer, pH 7, and pelleted by centrifugation. The cells are resuspended in 30-40 ml of buffer II and passed twice through a French pressure cell at a pressure in excess of 12,000 psi into a cold receptacle. The resulting suspension is centrifuged and dialyzed as described for sonication. Streptomycetes are resuspended in a minimum quantity of buffer ][, sonicated for 15 sec to disperse mycelial clumps and treated as described above. Enzymic Lysis by Lysozyme. Escherichia coli can be lysed by resuspending a washed cell pellet in 3 ml (per gram) of 0.1 M K~PQ (pH 7.5 at room temperature) containing 0.5 mg/ml EDTA and 1 mg/ml egg white lysozyme and incubating for 1 hr at room temperature. The resulting suspension is then centrifuged and dialyzed as described above. Lysozyme may also be used to lyse streptomycetes as described by Walker and Walker. 11 Frozen 2.5-day mycelial pads are dispersed and digested for 1 hr at room temperature in 3 volumes of 0.1 M potassium phosphate buffer (pH 7.4) containing 5 mg/ml EDTA and 1 mg/ml lysozyme. The supernatant is centrifuged at 30,000 g for 30 min at 4 °, dialyzed against 1 mM phosphate buffer-EDTA containing 0.1 ml fl-mercaptoethanol per 4 liters and finally dialyzed against 25 mM Tris (pH 7.4) containing fl-mercaptoethanol. A similar procedure has been described by Hey and Elbein. 12 Enzymic Lysis by Lysostaphin. Staphylococci are refractory to the action of lysozyme but can be lysed with lysostaphin. The washed cell pellet from a 100-ml early stationary phase culture is resuspended in 4 ml of buffer I, lysostaphin is added to a final concentration of 50 ~g/ml 1, j. B. Walker and M. S. Walker, Biochemi, try 6, 3821 (1967). ~ A. Hey and A. D. Elbein, J. Bacteriol. 96, 105 (1968).
622
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
and the mixture is incubated for 30 min at 37 °. The suspension is centrifuged and dialyzed as described for sonication. P o l y m y x i n B. Polymyxin treatment may be used to release aminoglycoside-modifying enzymes from E. coli '~ and some strains of Pseudomonas. A culture growing exponentially in glycerol-salts medium (per liter: 2.5 g glycerol, 7 g K2HPO4-3H20, 3 g KH~P04, 1 g (NH4)2SO~, 0.1 g MgSO4.7H~O, 0.5 g sodium citrate.5.5H.~O, 0.1 ml of a 0.335% solution of ferric citrate, and 0.1 ml of a 0.163% solution of MnS04, pH 7.0-7.2) is harvested by centrifugation at an A~oo of 1.0. The cells are washed once in one-third culture volume of 0.14 M NaC1, pH 7.3, centriftLged, resuspended in one-tenth of the original culture volume of 0.14 M NaC1 (pH 7.3) and treated for 1 min at 37 ° with 200 ~g/ml polymyxin B sulfate (Sigma). The residue is pelleted by centrifugation at 10,000 g or by filtration through cellulose acetate filters (pore size 0.45 ~m). The filtrate or supernatant contains the aminoglycoside-modifying enzymes. This technique has been reported to release periplasmic enzymes with a 50-100% yield while releasing only about 5% of the total cellular proteinJ 3 Purification
Despite several efforts, using many different techniques, 1~-1~ there is no satisfactorily documented account of the successful purification of any of the enzymes to homogeneity. In a number of cases, however, partial purification has been achieved, 17,18 usually by a combination of streptomycin sulfate and ammonium sulfate precipitations followed by DEAE-cellulose chromatography. In general, all operations are carried out at 4 ° . The normal starting point for partial purification is an osmotic shockate or a whole-cell lysate S100. Streptomycin sulfate is added to the cold enzyme solution to a final concentration of 1.5%. After the streptomycin has dissolved, the mixture is mixed gently for 15 rain and then centrifuged at 15,000 g for 15 rain. The enzyme is precipitated from the supernatant by a suitable ammonium sulfate cut (this usually lies between 30 and 60% saturation with ammonium sulfate); the precipitate from the final cut is resuspended in and dialyzed against a suitable buffer and absorbed 13G. Cerny and M. Teuber, Arch. Mikrobiol. 78, 166 (1971). 1'O. Doi, S. Kondo, N. Tanaka, and H. Umezawa, J. Antibiot. 22, 273 (1969). ~Y. Sakagami, N. Takaishi, and A. Hachimori, J. Antibiot. 27, 248 (1974). ~"M. J. Haas, unpublished data. 17A. L. Smith and D. H. Smith, g. Inject. Dis. 129, 391 (1974). ~8M. Brzezinska and J. Davies, Antimicrob. Ag. Chemother. 3, 266 (1973).
[48]
AMINOGLYCOSIDE-M ODIFYING ENZYMES
603
to a DEAE-eellulose column. The column is washed with a small volume of buffer, and protein is eluted with buffer containing a linear sodium chloride, ammonium chloride, or potassium acetate concentration gradient from 0 to 0.5 M. Such a procedure usually results in a 25-70% increase in specific activity with a recovery of 3o-7o% of the initial activity.
Properties General Properties. The aminoglycoside-modifying enzymes are poorly characterized from a physical and chemical point of view. Most of the enzymes are inactivated by repeated freezing and thawing and are stable at --70 °. Substrate inhibition is commonly observed. The molecular weights of several of the enzymes have been estimated at 20,000-30,000. TM In general, a suitable buffer for dialysis, storage and assay contains 10 mM Tris-ehloride or acetate, 10 mM magnesium chloride or acetate, 25 mM ammonium chloride, and a sulfhydryl reagent (DTT, 0.5 raM-1 mM or fl-mercaptoenthanol, 1 mM-3 mM), pH 7.0-8.0. Kanamgcin AcetyltransJeq'ase (E. coli R5/~:6771';; NR79/W677 ~) Stability and Activity. In unbuffered osmotic shockates the enzyme loses 8% of its activity during 4.5 hr of incubation at 30 °. The presence of 5-25% glycerol increases stability but decreases activity by as much as 30%. The enzyme is stable at --70 ° and is stable to lyophilization and acetone precipitation. The pH optimmn for the acetylation of the kanamycins, neomycins, and tobramycin is approximately 5.8; the optimum for gentamicins C1 and C1,~is near 7.6. Enzyme stability and activity are reduced by concentrations of DTT above 5 mM. EC]ect o] BuJ~ers and Ions. The standard buffcr is buffer III. Tricine, MOPS, and cacodylate buffers at 10 mM are satisfactory. In phosphate, HEPES, Kolthoff-borate-phosphate, and Tris-maleate buffers the enzyme is less stable. The enzyme requires 10 mM magnesimn for activity but is inhibited by concentrations above 20 raM. The enzyme is unstable in sodium chloride but is stable in potassium acetate. Kinetic Data. The Kn, for the aeetylation of kanamyein A is 1 ~M at pH 7.0. The V..... is 1 ~mole/min using the assay conditions described. The acetylation of kanamyein is inhibited by paromomyein, paromamine, and gentamiein A. Substrate inhibition occurs at drug concentrations above 0.1 raM. ' ' J . Davfi~s and R. Benvcniste, Ann. N . Y . Acad. Sci. 235, 130 (1974).
624
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
Kanamycin Acetyltrans]erase (P. aeruginosa 37962°) Stability. KAT-3796 is stabilized by glycerol and ammonium chloride. The standard working buffer is buffer IV. Activity. The pH optimum for the acetylation of kanamycin A is between 5.6 and 6.5, and no pH optimum is exhibited between 5 and 9 for the acetylation of sisomicin and gentamicin C1~. The acetylation of kanamycin A, neomycin B and gentamicin Cla is inhibited by kanamycin C, gentamicin A, and, to a lesser extent, lividomycin.
Gentamicin Acetyltrans]erase I (E. coli R135/C6002°) Stability. The enzyme is stable at --20 ° and rapidly loses activity above 35 °. It is generally less stable than a similar enzyme isolated from P. aeruginosa 130. The standard buffer is buffer V. Kinetic Data. The Km for the acetylation of sisomicin is approximately 10 pjl//, and the V~ax is 0.9 nmole/min. The enzyme is inhibited by concentrations of sisomicin (but not of gentamicin C1) above 0.1 raM. Gentamicin Acetyltrans]erase I (P. aeruginosa 1302°) Stability. The enzyme is stable at --20 ° and is rapidly inactivated above 35 °. Buffer V is the standard buffer. pH Optimum. The pH optimum for the acetylation of sisomicin and kanamycin B is about 7.4. Gentamicin C1 acetylation exhibits a broad pH optimum (pH 5.4-8.0). Kinetic Data. The K,, for sisomicin acetylation is approximately 0.1 mM and the V~ax is about 60 nmole/min. The enzyme is inhibited by sisomicin, but not gentamicin C~, at concentrations above 0.1 mM. Gentamicin Acetyltrans]erase II (Providencia 16421) Stability. The enzyme is stable at --20 ° . Reactions are routinely supplemented with magnesium acetate and DTT although an absolute requirement for these has not been demonstrated. Activity. The pH optimum for acetylation of kanamycin C is 6.0; for gentamicin C1 it is 6.6. The enzyme is inhibited by poor substrates and nonsubstrates. 20S. Biddlecome, M. S. thesis, University of Wisconsin,Madison, 1973. 51M. Chevereau, P. J. L. Daniels, J. Davies, and F. LeGoffic, Biochemistry 13, 598 (1974).
[48]
AMI NOGLYCOSIDE-MODIFYING ENZYMES
625
Gentamicin Acetyltrans]erase III (P. aeruginosa PST-1 ~°) Stability. The enzyme is stable at --20 ° . It is not inactivated by incubation at 30 ° for 20 min. Activity. The pH optimum for the acetylation of the kanamycins, gentamicins, and neomycins is between pH 5.5 and 6.0. Magnesium (10 mM optimum) and a sulfhydryl reagent (DTT, 3 mM optimum) are required for activity but not for stability. Substrate inhibition occurs at varying concentrations with different substrates. Gentamicin A denylytrans]erase (E. coli JR66/W67722,-~:~) Requirements Jor Activity. DTT (1-10 mM) and magnesium are necessary for the maintenance of activity. Removal of magnesium results in irreversible inactivation. The enzyme is inhibited by high salt concentrations. pH Optimum. The pH optimum for the adenylylation of the gentamicins and kanamycins is 8.0-8.2. Streptomycin-Spectinomycin AdenylyltransJerase (E. coli NR73/W67724) Stability and Activation. The enzyme is stable at --10 ° and is activated by concentrations of ammonium chloride above 1 M. Ion and Co]actor Requirements. For the maintenance of activity and stability, 10 mM magnesium is required and may be replaced by manganese (0.1 mM), zinc, or cadmium. The enzyme will use ATP or dATP as cofactors. Kinetic Data. The Km's for the adenylylation of streptomycin and spectinomycin are 20 t~M. Activity is 50% reduced by 30 t~M pyrophosphate, but not by azide, iodide, cyanide, or pCMB. The enzyme is inhibited by streptomycin or spectinomycin at 0.2 mM. Streptomycin adenylylation is inhibited by tetracycline. Streptomycin-Spectinomycin AdenylyltransJe,'ase (E. coli B/RE130 '-'~) Activity Requirements. The enzyme requires magnesium (8-10 mM) and a reducing agent for maximal activity. Magnesium cannot be re52R. Benveniste, Ph.D. thesis, University of Wisconsin, Madison, 1972. 53M. Yagisawa, H. Naganawa, S. Kondo, M. Hamada, T. Takeuchi, H. Umezawa, J. Antibiot. 24, 911 (1971). 2~R. Benveniste, M. S. thesis, University of Wisconsin, Madison, 1970. 25j. H. Harwood and D. H. Smith, J. Bacteriol. 97, 1262 (1969).
626
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
placed by nickel, cobalt, zinc, calcium, or manganese. The pH optimum for the adenylylation of streptomycin is near 8.3. The enzyme will use ATP and dATP as cofactors.
Neomycin Phosphotrans]erase I (E. coli JR35/W677:6,27; JR39/W6772s; ML162929)
Activity and Stability. The enzyme from JR39 has an absolute requirement for magnesium and is stabilized by 0.1 M ammonium chloride or 10 ~g/ml neomycin B. The enzyme from ML1629 is inactivated by 5 min of incubation at 45 °. It also requires magnesium. DTT, 10 mM, prevents denaturation and restores the activity of denatured enzyme. Inhibition. Phosphorylation of neomycin B is inhibited by gentamicin C1~ and by tobramycin. Neomycin Phosphotrans]erase H (E. coli JR66/W677~s,29; P. aeruginosa Ps49 ~8) Stability. The enzyme is stabilized by 10 ~g/ml neomycin B or 0.1 M ammonium chloride. Activity. There is no marked pH optimum between pH 5.5 and 8.0 for the phosphorylation of neomycin B. Neomycin B phosphorylation is not inhibited by tobramycin or by gentamicin C1~ but phosphorylation of kanamycin C is inhibited by tobramycin. Neomycin PhosphotransJerase (P. aeruginosa H-93°) Stability. The phosphorylation of kanamycin proceeds for 20 rain at 55 °, for 5 min at 65 ° and not at all at 75% pH and heat stability are not markedly affected by 10 mM EDTA, 10 mM magnesium acetate, or 0.4 mM kanamycin. The enzyme is stable between pH 7.5 and 9. pH and Kinetic Data. The pH optimum for the phosphorylation of kanamycin is 7.5. At this pH, the Km for the phosphorylation of kanamycin is 0.3 raM, and the Vm~xis 42 ~moles/hr/OD unit. Ion and Cofactor Requirements. The enzyme requires magnesium, manganese, zinc, or cobalt (all at 1 raM) for activity. ATP is the only ~ M. S. Okanishi, S. Kondo, R. Utahara, and H. Umezawa, J. Antibiot. 21, 13 (1967). 21B. Ozanne, R. Benveniste, D. Tipper, and J. Davies, J. Bacteriol. 100, 1144 (1969). M. Brzezinska and J. Davies, Antimicrob. Ag. Chemother. 3, 266 (1973). 20M. Yagisawa, H. Yamamoto, I-I. Naganawa, S. Kondo, T. Takeuchi, and H. Umezawa, J. Antibiot. 25, 748 (1972). 3, O. Doi, S. Kondo, N. Tanaka, and H. Umezawa, J. Antibiot. 22, 273 (1969).
[48]
AMINOGLYCOSIDE-MODIFYING ENZYMES
627
accepted phosphoryl donor when partially purified enzyme preparations are used; crude preparations will use other nucleoside triphosphates. One hundred percent of the available kanamycin is modified in the presence of equimolar ATP.
Neomycin Phosphotrans/erase (S. aureus B294 :~) pH Optimum. The pH optimum for the inactivation of kanamycin is between pH 7.0 and 7.5. Inhibition. Substrate inhibition occurs at a kanamycin concentration of 2 m M but not at 0.2 mM. Lividomycin Phosphotrans/erase (E. coli R,,~+/ML14103'-'; P. cleruginosa Ti-1333; S. aureus KW-234)
Activity Requirements. The three enzymes require A T P and magnesium for activity.
pH Optimum. The pH optimum for the phosphorylation of lividomycin is between 6.5 and 7.0 for the three enzymes. Streptomycin Phosphotrans/erase (E. coli JR35/W677 '-,T) Ion and Co/actor Requirements. Magnesium is required for maximal activity, but not for stability, and can be partially replaced by zinc or manganese but not by copper (all at 5 raM). The enzyme uses A T P or G T P (but not C T P or U T P ) . pH Optimum. The pH optimum for the phosphorylation of strei)tomycin is 8.0. Streptomycin Phosphotransferase (P. aeruginosa H-93~) pH Optimum. The pH optimum for the phosphorylation of streptomycin is 8.5.
Co/actor Requirements. The enzyme will use ATP, CTP, GTP, or U T P ~' O. Doi, M. Miyamoto, N. Tanaka, and H. Vmezawa, Appl. Microbiol. 16, 1282 (1968). 3;M. Yamaguchi, T. Koshi, F. Kobayashi, and S. Mitsuhashi, Antin~icrob. Ag. Chemother. 2, 145 (1972). ~F. Kobayashi, M. Yarnaguchi, and S. Mitsuhashi, Antimic~'ob. Ag. Chemother. 1, 17 (1972). ~ F. Kobayashi, T. Koshi, J. Eda, Y. Yoshimura, and S. Mitsuhashi, Antimicrob. Ag. Chemother. 4, 1 (1973). '~ O. Doi, M. Ogura, N. Tanaka, and H. Umezawa, Appl. Microbiol. 16, 1276 (1968).
628
[49]
ANTIBIOTIC INACTIVATION AND MODIFICATION
as cofactors; maximal activity is observed at an ATP concentration of 2O mM. Acknowledgment We thank Dr. Julian Davies for many useful contributions to this work.
[49] ATP:Streptomycin 6-Phosphotransferase: By JAMES B. WALKER Dihydrostreptomycin +
MgATP
'l
NH,
MgADP
L~---o ,\
.,c~/, OH
C:NH + I NH
+
OH
I
J - - - - t ~,=H~
, "Kc.,o. I
NH z
/,'-O.~oH
,
(1)
)~
oPO;-
I
Dihydrostreptomycin-6-P
H~N--C--NH
Streptidine
+
MgATP
,
NH2 I + C=NH,
~ MgADP
(2) tt
oPofSt reptidine - 6 - P
Streptomycin 6-kinase is present in high concentration in extracts of streptomycin-producing strains of Streptomyces. 2-~ This enzyme can also 1EC 2.7.1.72 (mistakenly called 5-kinase). "J. B. Walker and M. S. Walker, Biochim. Biophys. Acta 148, 335 (1967). 3 A. L. Miller and J. B. Walker, J. BacteTiol. 99, 401 (1969). , M. S. Walker and J. B. Walker, J. Biol. Chem. 245, 6683 (1970). O. Nimi, G. Ito, Y. Ohata, S. Funayama, and R. Nomi, Agr. Biol. Chem. 3~, 850 (1971).
AMINOGI.,YCOSIDE-MODIFYING ENZYMES
611
[48] Aminoglycoside-Modifying Enzymes B y MICHAEL J. HAAS and JOHN E. DOWDING
This article describes the isolation and assay procedures currently used in the study of the nine aminoglycoside-modifying activities listed in the table. It should be emphasized that very little information is available for some of the enzymes listed in the table and for this reason independently isolated activities catalyzing the same reaction have been grouped together (e.g., the three kanamycin acetyltransferases) pending studies showing them to be substantially different enzymes. These enzymes, which modify the aminoglycoside or aminocyclitol antibiotics, have been detected in a wide variety of resistant bacteria. In many clinical isolates they are known to be plasmid-coded, and in certain strains the enzymes appear to be located near the cell surface) a fact which is used to advantage in some of the enzyme isolation techniques described below. Among Eubacteria the three known aminoglycoside-modification mechanisms are acetylation of amino groups and phosphorylation and adenylylation of hydroxyl groups; these mechanisms have recently been reviewed by Benveniste and Davies. 1 Other modified compounds have been detected among antibiotic-producing organisms,2, 3 and it is possible that in these strains these may be biosynthetic intermediates or inactivation products. The structures of the aminoglycoside antibiotics (with positions of modification) are shown in Figs. 1-7. Although an indication of the modification site may often be obtained by examination of the substrate specificity of an enzyme, it has been determined in most cases by chemical and/or spectroscopic analysis of the modified antibiotic (table). The study of these enzymes is particularly valuable for two reasons. First, determinations of the type and site of modification have allowed the design and synthesis of semisynthetic antibiotics not modified by the enzyme and therefore active against many resistant isolates. Second, the enzymes provide a very sensitive, rapid and often specific assay for the aminoglycoside antibiotics2, ~ It is important to remember that many R ]actor-containing strains
1R. Benveniste and J. Davies, Annu. Rev. Biochem. 42, 471 (1973). "~M. K. Majumdar and S. K. Majumdar, Biochem. J. 120, 271 (1970). M. Kojuma, S. Inouye, and T. Niida, J. Antibiot. 26, 246 (1973). 4j. Davies, M. Brzezinska, and R. Benveniste, Ann. N.Y. Acad. Sci. 182, 226 (1971), 5M. J. Haas and J. Davies, Antimicrob. Ag. Chemother. 4, 497 (1973).
612
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
/b HO,--x--.~C,H,~O\
o"
I- b
d
7
~
C H o ~ N H R
3
0
~o~H~ H O ~
Kanamycin A Kanamycin B Kanamycin C BB-K8 (amikacin)
Rl
R~
NH~ NH~ OH NH~
OH NH2 NH: OH
OH OH R3
H H H --CO---CH(OH)CH:--CH2NH~
Fro. 1. Structure of the kanamycins. Arrows indicate sites of O-phosphorylation (a), N-acetylation (b, c, d), and O-adenylylation (e).
are pathogens or potential pathogens. Care should be exercised when handling cultures and all used media and glassware should be sterilized be]ore disposal or cleaning.
Assay Procedures General Protocol. Enzyme activities are assayed by means of the phosphocellulose paper binding assay devised by Davies et al. 4 which measures transfer of radiolabel from a suitable cofactor to the antibiotic. The general assay procedure used is outlined below and is followed by details for each type of reaction. The reaction mixture consists of buffer, labeled cofactor, enzyme prepa s
I
0 NH,7
~
NH~
O OH
t
d
Fxo. 2. Structure of tobramycin (nebramycin factor 6). Arrows indicate sites of N-acetylation (a, b, c) and O-adenylylation (e).
[48]
AMINOGLYCOSIDE-MODI FYI NG ENZYMES
.R, -
613
/b
S(~H-R~,-'I~O \
d
o R~
/
e
Gentamicin Gentamicin Gentamicin Gentamicin Gentamicin
A B CI~ C~ C1
R1
R~
R3
R4
R5
R6
R7
H H H CH3 CHa
OH NH2 NH~ NH~. NHCHa
OH H H It H
OH OH H H H
NH2 OH NH2 NH~ NH..
H OH OH OH OH
OH CHa CHa CH3 CH3
FxG. 3. Structure of the gentamicins. Sisomicin is 4,5-dehydrogentamicin C1, (ring I). Arrows indicate sites of O-phosphorylation (a), N-acetylation (b, c, d), and O-adenylylation (e). a r a t i o n a n d a n t i b i o t i c . A c r u d e e n z y m e e x t r a c t (S100 or o s m o t i c shocka t e ) is of s u i t a b l e p u r i t y for t h e a s s a y . F o r large n u m b e r s of a s s a y s it is c o n v e n i e n t to d i s t r i b u t e p r e m i x e d buffer, c o f a c t o r , a n d e n z y m e (or buffer, cofaetor, a n d a n t i b i o t i c ) . Such m i x t u r e s s h o u l d n o t be r e - u s e d b
HO
6CHL'N/H O.
R3
Ribostamycin Butirosin A Butirosin B
OH
R,
1{2
R3
H - - C O--C H (O H) - - C H r--C H..,NH.., --CO--CH(OH)--CH2--CH~NH2
H OH H
OH H OH
FIG. 4. Structures of ribostamycin and the butirosins. Arrows indicate sites of O-phosphorylation (a) and N-acetylation (b, c, d).
614
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
/b 0 ,-.~,^
c
o"7" ~ - - - ~ --.~'. %
I).o\?-..y6.
'~ '2
R,~
Rl
R~
Neomycin B Neomycin C
NH2 NH2
OH OH
Paromomycin
OH
OH
Lividomycin A Lividomycin B
OH OH
H H
0
---~-
=
OH
R8
R4
H CH2NH2 H + {CH.NH~ H H
R~
CH~NH2 H
H H
CH~NH2}
H
CH2NH2 CH:NH2
mannose H
FIe. 5. Structures of the neomycins and lividomycins. Neamine (and paromamine) are rings I and II. Arrows indicate sites of O-phosphorylation (a) and N-acetylation (b, c, d). NH II HNCNH2
NH CNH2
0
H
R~
OH
Ii"
OH ~) R=O C~H~HO4 /OH
Streptomycin Dihydrostreptomycin Mannosidostreptomycin
RI
R2
CHO CH2OH C HO
H H mannose
Fro. 6. Structure of the streptomycins. Ring I is streptidine, ring II is streptose, and ring III is N-methyl4.-glucosamine. The arrow indicates the site of O-adenylylation or phosphorylation.
[481
AMINOGLYCOSIDE-MODIFYIN G ENZYMES
~
()l;~
CH3
0 FIG. 7. Structure of speetinomycin. The arrow indicates the site of O-adenylylation. after refrigeration or freezing. Control (blank) assays lacking either antibiotic or enzyme should be included in each set of assays. The components of the assay are mixed in chilled 75 X 10 mm tubes and the reaction is started by incubation at 30 ° or 35 °. After 15-20 rain of incubation the tubes are returned to the ice bath and a 10-20 ~1 sample of each reaction mixture is pipetted onto a numbered piece (ca. 1 cm ~) of W h a t m a n P-81 phosphocellulose paper raised above a block of styrofoam on a pin. After 30 sec at room temperature papers and pins are placed in about 500 ml of hot (70--80 °) distilled water for 3-4 min. The liquid, which contains unused labeled cofactor, is poured off and disposed of accordingly. The papers are rinsed 3 or 4 times with 500-600 ml of distilled water and dried under heat lamps (avoid charring) or using a hot-air dryer. Papers (with or without pins) are placed in glass scintillation vials containing 10 ml of toluene scintillation fluid (3 g PP(), 0.1 g P O P O P per liter of toluene) and counted. A time course of inactivation may be determined by simply scaling up the reaction mixture and removing samples for counting at various times. Descriptions of large-scale inactivations for chemical analyses m,~y be found among the references in the table. Assay o.f Kanamycin and Gentamicin Acetyltrans]erases. Acetyltransferases with acid pH optima are assayed in a buffer prepared by adding to 30 ml of deionized water: 12.75 ml of 0.6 M citric acid, 27.25 rnl of 0.6 M tripotassium citrate, 2.4 ml of 1.0 M magnesium acetate, and 1.6 ml of 0.5 M D T T . The pH is adjusted to the desired value at 30 ° with potassium hydroxide or glacial acetic acid and the volume is adjusted to 80 ml with deionized water. Acetyltransferases with neutral or slightly basic pH optima are assayed in a buffer prepared by adding to 40 ml of deionized water: 25 ml of 1 M Tris base, 5 ml of 1 M MgCl~, 1 ml of 0.5 M D T T and 10 ml of 1 M NH4C1. The pH is adjusted to the desired value at 30 ° with HC1 or glacial acetic acid and the volume is adjusted to 100 ml with deionized water.
616
ANTIBIOTIC INACTIVATION AND MODIFICATION A M I N O G L Y C O S I D E - M ODIFYING
Enzyme-
[48]
ENZYMES
Synonyms
Substrates b
Kanamycin acetyltransferase (KAT)
--
Gentamicin acetyltransferase I (GATI)
--
Neomycins, kanamycins A and B, gentamicin Cla, tobramycin, butirosins, ribostamycin, sisomicin, BB-K8 (gentamicin C2) Gentamicins, sisomicin (kanamycin B, tobramycin)
Gentamicin acetyltransferase II (GATH)
Gentamiein acetyltransferase I I I (GATm) Gentamicin adenylyltransferase (GAdT) Streptomycin-spectinomycin adenylyltransferase (SAdT) Neomycin phosphotransferase I (NPTI) Neomycin phosphotransferase II (NPT~) Streptomycin phosphotransferase (SPT)
Gentamicin adenylate synthetase (GAS), kanamyein nucleotidyltransferase (KNT) k Streptomycin adenylate synthetase (SAS) Kanamyain phosphotransferase I (KPTI) n, possibly also lividomycin phosphotransferase (LPT) Kanamycin phosphotransferase II (KPTII) n
Kanamycin C, gentamicins, sisomicin, tobramycin, butirosins (neomycins, ribostamycin kanamycin B) Kanamycins, gentamicins, sisomicin, ribostamycin, tobramycin, lividomycins Kanamycins, gentamicins, tobramycin Streptomycin, spectinomycin, dihydrostreptomycin (mannosidostreptomycin) Neomycins, kanamycins, lividomycins, ribostamycin, gentamicins A and B Neomycins, kanamycins, butirosins, ribostamycin, gentamicins A and 'B Streptomycin, dihydrostreptomycin
"Enzyme names and abbreviations are currently under review. b Poor substrates are shown in parentheses. c Aminoglycoside structures are shown in Figs. 1-7. d H. Umezawa, M. Okanishi, R. Utahara, K. Maeda, and S. Kondo, J. Antibiot. 20, 136 (1967). M. Yagisawa, H. Naganawa, S. Kondo, T. Takeuchi, and H. Umezawa, J. Antibiot. 25, 495 (1972). I R. Benveniste and J. Davies, Proc. Nat. Acad. Sci. U.S. 70, 2276 (1973). g M. Brzezinska, R. Benveniste, J. Davies, P. J. L. I)aniels, and J. Weinstein, Biochemistry 11, 761 (1972). h M. Chevereau, P. J. L. Daniels, J. Davies, and F. Le Goffic, Biochemistry 13, 598 (1974). i S. Biddlecome, P. Daniels, J. Davies, M. Haas, and D. Rane, in preparation. i H. Naganawa, M. Yagisawa, S. Kondo, T. Takeuchi, and H. Umezawa, J. Antibiot. 24, 913 (1971).
[48l
AMINOGLYCOSIDE-MODIFYING ENZYMES
Cofactors
Modificationc
617
Representative strains ~
Acetyl coenzyme A
6-Amino group of aminohexose I is aeetylated a,e,/
Acetyt coenzyme A
3-Amino group of deoxystreptamine (II) is acetylatedg
Acetyl coenzyme A
2-Amino group of aminohexose I is acetylated/,h
Acetyl coenzyme A
3-Amino group of deoxystreptamine (II) is acetylated ~
P. aeruginosa PST 1*
Ribo- or deoxyribonucleoside triphosphates (ATP preferred)
2-Hydroxyl group of aminohexose I I I is adenylylatedi
E. coli JR66/W677
ATP or dATP m
3-Hydroxyl group of Nmethyl-I~glucosamine is adenylylated z 3-Hydroxyl group of aminohexose I ° or 5-hydroxyl group of pentose I I I ~ is phosphorylated 3-Hydroxyl group of aminohexose I is phosphorylated
E. coli RlOO/W4354," NR73/W677
ATP
ATP ATP
3-Hydroxyl group of Nmethyl-L-glucosamine is phosphorylated~
E. coli R5/W677~; NR79/ W677, P. aeruginosa GN315/3796, Streptomyces kanamyceticus ATCC 12853/ P. aeruginosa 1 3 0 / P . aeruginosa 2 0 9 / E . coli R135/C600 Providencia 164,h Streptomyces spectabilis UC 2472/
E. coli JR35/W677p
E. coli JR66/W677,q P. aeruginosa Ps49q E. coli J R 3 5 / W 6 7 7 / P. aeruginosa H-9, r Staphylococcus aureus B294," Streptomyces griseus ATCC 10971t
k S. Kondo, K. Iinuma, M. Hamada, K. Maeda, and H. Umezawa, J. Antibiot. 27, 90 (1974). z T. Yamada, D. Tipper, and J. Davies, Nature (London) 219, 288 (1968). " R. Benveniste, T. Yamada, and J. Davies, Infection Immunity 1, 109 (1970). " H. Umezawa, H. Yamamoto, M. Yagisawa, S. Kondo, T. Takeuchi, and Y. A. Chabbert, J. Antibiot. 26, 407 (1973). ° S. Kondo, M. Okanishi, R. Utahara, K. Maeda, and H. Umezawa, J. Antibiot. 21, 22 (1968). p B. Ozanne, R. Benveniste, D. Tipper, and J. Davies, J. Bacteriol. 100, 1144 (1969). q M. Brzezinska and J. Davies, Antimicrob. Ag. Chemother. 3, 266 (1973). r O. Doi, M. Ogura, N. Tanaka, and H. Umezawa, Appl. Microbiol. 16, 1276 (1968). O. Doi, M. Miyamoto, N. Tanaka, and H. Umezawa, Appl. Microbiol. 16, 1282 (1968). t j. Walker and M. Skorvaga, J. Biol. Chem. 248, 2435 (1973). " Cultures not available from culture collections may usually be obtained from authors of relevant publications.
618
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
Lyophilized [1-14C]acetyl coenzyme A (50-60 Ci/mole) is resuspended in deionized water to an activity of 25 ~Ci/ml and diluted with 2 parts of 0.88 mg/ml trilithium acetyl coenzyme A before use. Each assay contains: 10 t~l of buffer, 10 t~l of [14C]acetyl-CoA solution, 5 ~l of enzyme preparation, 5 ~l of antibiotic (1 mg/ml). The mixture is incubated at 30 ° for 15 min and 20-~1 samples are counted. If diluted or less active enzyme preparations are being assayed, up to 20 t~l may be used in each assay. Assay o] Neomycin and Streptomycin Phosphotrans]erases. To prepare 30 ml of buffer (sufficient for 3000 assays), mix 15 ml of distilled water with 2 ml of 1 M Tris, 1.25 ml of 1 M MgCl~, 6 ml of 2 M NH4CI, and 0.1 ml of 0.5 M DTT. Adjust the pH to 7.1 at 5 ° with 1 M maleic acid and make up to 30 ml with deionized water. [~2P]ATP solution consists of 15 ~l of 50 mM sodium ATP (adjusted to pH 7.2 with NaOH) and 10-100 ~1 of [~/-~P]ATP depending on specific activity (initially-~ 20 Ci/mole, 0.75 mCi/ml) made up to 1 ml with deionized water. Each assay contains: 10 ~l of buffer, 10 ~l of [3-°P]ATP solution, 10 t~l of enzyme preparation, 2 ~l of antibiotic (1 mg/ml). The mixture is incubated at 35 ° for 20 min, and 20-~1 samples are counted. COMMENTS. Owing to the short half-life of 32p, ATP solutions should be used within 2-3 weeks of preparation. Assays o/ Gentamicin and Streptomycin Adenylyltrans]erases. The assay buffer is the same as for phosphotransferase assays except that it is brought to pH 7.8 at 5 °. Uniformly labeled [~C]ATP (400-500 Ci/mole) is purchased in 50% ethanol, lyophilized to dryness and resuspended in deionized water to an activity of 50 ~Ci/ml. To 1 ml of this are added 50 ~l 0.1 M ATP and 3.95 ml of deionized water, yielding a final cofactor preparation of approximately 10 t~Ci/tLmole/ml. Each assay contains: 10 ~l of buffer, 10 ~l of [~4C]ATP solution, 10 ~l of enzyme preparation, 2 ~l of antibiotic (1 mg/ml). The mixture is incubated at 30 ° for 30 min and 20- or 25-~1 samples are counted. COMMENTS. [14C]ATP may be purchased in 2% ethanol, when it is used directly after dilution. [a-32P]ATP may also be used as an adenyl donor but the short half-life of 32p is usually a disadvantage. A number of other methods have been used to assay inactivation by aminoglycoside-modifying enzymes, the most common being a microbiological assay which measures residual antibiotic activity by disc assay against a sensitive test organism2 Although this assay is convenient and T. Gavan, E. Cheatle, H. McFadden, Jr., Eds., "Antimicrobial Susceptibility Testing." American Society of Clinical Pathologists, Chicago, Illinois (1971).
[481
A M I N O G L Y C O S I D EODIFYI -M NG ENZYMES
619
simple, it may not be sufficiently quantitative for some experiments (e.g., time courses of inactivation). In addition, it cannot measure modification of a compound that is not an antibiotic. Furthermore, modification may not be detected if it does not result in complete inactivation (e.g., 6'-Nacetylneomycin B retains considerable antimicrobial activity). N-Acetylation may be monitored by means of a colorimetric assay using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). 7 Free eoenzyme A formed during acetylation reacts with DTNB to form a mixed disulfide; this is detected by an increase in A41_~. The assay is useful for certain antibiotics which do not bind quantitatively to phosphocellulose paper or if radiolabeled acetyl CoA is unavailable, but it requires the use of at least partially purified enzyme preparations. Sulfhydryl reagents interfere with the DTNB assay. The products of modification reactions may also be identified by cellulose-acetate strip electrophoresis 7 and thin-layer chromatography ;~ residual antibiotic can be determined by high-pressure liquid chromatography, s Growth of Cells
Drug-resistant bacteria are grown at 37 ° in medium containing, per liter: 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride. Ten grams of glucose are added separately, as a sterile 50% solution, after sterilization of the medium. One of the antibiotics to which the R factor confers resistance is routinely added to the medium after sterilization (to a final concentration of 20 ~g/ml) to prevent possible segregation of the R factor. Streptomycetes are grown for 18-36 hr at 30 ° in Difco Nutrient Broth supplemented after sterilization with glucose (final concentration 1%).
Buf]ers Buffer I: 10 mM Tris-chloride, 10 mM magnesium chloride, 25 mM ammonium chloride, 0.6 mM fl-mercaptoethanol, pH 7.8 at 4 ° Buffer II: 20 mM Tris-chloride, 10 mM magnesium acetate, 25 mM ammonium chloride, 10 mM potassium chloride, 2 mM DTT, pH 7.5 at 4 ° Buffer III: 10 mM Tris-acetate, 10 mM magnesium acetate, 1 mM fl-mercaptoethanol, pH 7.4, at 4 °. pH is adjusted with acetic acid. Buffer IV: 20 mM Tris-chloride, 10 mM magnesium chloride, 10 mM potassium chloride, 30% glycerol, pH 7.8 R. Benveniste and J. Davies, Biochemistry 10, 1787 (1971). s H. Umezawa, H. Yamamoto, M. Yagisawa, S. Kondo, T. Takeuchi, and Y. Chabbert, J. Antibiot. 26, 407 (1973).
620
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
Buffer V: 10 mM Tris-chloride, l0 mM magnesium chloride, 3 mM, fl-mercaptoethanol, pH 7.8
Isolation of Crude Enzyme Preparations For those species (Escherichia, Shigella, Enterobacter, Serratia, Salmonella, and some strains of Pseudomonas) which release their drugmodifying activity when subjected to an osmotic shock, the procedure of Neu and Heppel 9 is used to obtain crude enzyme preparations. This technique solubilizes essentially all the aminoglycoside activity of an R ÷ E. coli while releasing only 4-6% of the total cell protein. One to two percent of the cells are lysed duriag such treatment. Where purification of an enzyme is desired, it is obviously an advantage if the R factor can be transferred to a strain susceptible to osmotic shock. 1° Aminoglycoside-modifying enzymes may be isolated from strains not susceptible to osmotic shock (notably Proteus, Providencia, Staphylococcus, Streptomyces, and some strains of Pseudomonas) either by sonication, French pressing, or enzymic lysis of cells. Osmotic Shock. Late log phase cells are harvested by centrifugation and resuspended in 100 volumes of 10 mM Tris-chloride, 30 mM sodium chloride, pH 7.8 at room temperature. The cells are again harvested by centrifugation, resuspended in 35 volumes of 3 mM EDTA, 33 mM Trischloride, pH 7.8, in 20% sucrose and stirred for 15 min at room temperature (a magnetic stirring bar is convenient). The cells are centrifuged at 16,000 g for 15 min in a refrigerated centrifuge, and the supernatant is removed. The centrifuge tube is inverted, and the pellet is allowed to drain in the cold for 10 min. It is important to remove all traces of sucrose or the cells will not shock properly. Therefore all excess fluid is removed from the cell pellet with cotton or other absorbent material. The ceils are resuspended in 40 volumes of ice-cold 0.5 mM m~gnesium chloride, stirred at 4 ° for 10-20 min and then centrifuged at 26,000 g for 30 min at 4 °. The pellet is discarded; the supernatant ("osmotic shockate") contains the aminoglycoside-modifying enzymes. Sonication. Washed log phase cells are resuspended in an appropriate buffer (0.6 g wet cells to 8 ml of buffer). The suspension, cooled in an ice bath, is subjected to 30-see bursts from a sonicator at 100 W output. Bursts are alternated with 2-min cooling periods, and 3-5 bursts are usually adequate (cell breakage may be monitored by phase-contrast microscopy). Some staphylococci cannot be broken by sonication; others may reH. C. Neu and L. A. Heppel, J. Biol. Chem. 240, 3685 (1965). 1, j. Davies, this volume [3].
[4sl
AMINOGLYCOSIDE-MODIFYING ENZYMES
621
quire the use of glass powder and longer sonication times for efficient breakage. Streptomycetes are resuspended in a minimum volume of Buffer I and sonicated as above. Sonicated cell suspensions are centrifuged at 30,000 g for 20-30 min, and the supernatant is dialyzed. The extract may be further clarified by preparing a 100,000 g supernatant (S100); deoxyribonuclease I is added to the 30,000 g supernatant to a final concentration of 4 ~g/ml, and the mixture is centrifuged at 100,000 g for 2 hr at 4 °. The supernatant is dialyzed against an appropriate buffer (usually buffer I or buffer II--see above). French Pressure Cell. The French pressure cell is the most convenient method of breaking large quantities of cells. The cells from 6 liters of a log phase culture are harvested by centrifugation, resuspended in 1 liter of 10 mM phosphate buffer, pH 7, and pelleted by centrifugation. The cells are resuspended in 30-40 ml of buffer II and passed twice through a French pressure cell at a pressure in excess of 12,000 psi into a cold receptacle. The resulting suspension is centrifuged and dialyzed as described for sonication. Streptomycetes are resuspended in a minimum quantity of buffer ][, sonicated for 15 sec to disperse mycelial clumps and treated as described above. Enzymic Lysis by Lysozyme. Escherichia coli can be lysed by resuspending a washed cell pellet in 3 ml (per gram) of 0.1 M K~PQ (pH 7.5 at room temperature) containing 0.5 mg/ml EDTA and 1 mg/ml egg white lysozyme and incubating for 1 hr at room temperature. The resulting suspension is then centrifuged and dialyzed as described above. Lysozyme may also be used to lyse streptomycetes as described by Walker and Walker. 11 Frozen 2.5-day mycelial pads are dispersed and digested for 1 hr at room temperature in 3 volumes of 0.1 M potassium phosphate buffer (pH 7.4) containing 5 mg/ml EDTA and 1 mg/ml lysozyme. The supernatant is centrifuged at 30,000 g for 30 min at 4 °, dialyzed against 1 mM phosphate buffer-EDTA containing 0.1 ml fl-mercaptoethanol per 4 liters and finally dialyzed against 25 mM Tris (pH 7.4) containing fl-mercaptoethanol. A similar procedure has been described by Hey and Elbein. 12 Enzymic Lysis by Lysostaphin. Staphylococci are refractory to the action of lysozyme but can be lysed with lysostaphin. The washed cell pellet from a 100-ml early stationary phase culture is resuspended in 4 ml of buffer I, lysostaphin is added to a final concentration of 50 ~g/ml 1, j. B. Walker and M. S. Walker, Biochemi, try 6, 3821 (1967). ~ A. Hey and A. D. Elbein, J. Bacteriol. 96, 105 (1968).
622
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
and the mixture is incubated for 30 min at 37 °. The suspension is centrifuged and dialyzed as described for sonication. P o l y m y x i n B. Polymyxin treatment may be used to release aminoglycoside-modifying enzymes from E. coli '~ and some strains of Pseudomonas. A culture growing exponentially in glycerol-salts medium (per liter: 2.5 g glycerol, 7 g K2HPO4-3H20, 3 g KH~P04, 1 g (NH4)2SO~, 0.1 g MgSO4.7H~O, 0.5 g sodium citrate.5.5H.~O, 0.1 ml of a 0.335% solution of ferric citrate, and 0.1 ml of a 0.163% solution of MnS04, pH 7.0-7.2) is harvested by centrifugation at an A~oo of 1.0. The cells are washed once in one-third culture volume of 0.14 M NaC1, pH 7.3, centriftLged, resuspended in one-tenth of the original culture volume of 0.14 M NaC1 (pH 7.3) and treated for 1 min at 37 ° with 200 ~g/ml polymyxin B sulfate (Sigma). The residue is pelleted by centrifugation at 10,000 g or by filtration through cellulose acetate filters (pore size 0.45 ~m). The filtrate or supernatant contains the aminoglycoside-modifying enzymes. This technique has been reported to release periplasmic enzymes with a 50-100% yield while releasing only about 5% of the total cellular proteinJ 3 Purification
Despite several efforts, using many different techniques, 1~-1~ there is no satisfactorily documented account of the successful purification of any of the enzymes to homogeneity. In a number of cases, however, partial purification has been achieved, 17,18 usually by a combination of streptomycin sulfate and ammonium sulfate precipitations followed by DEAE-cellulose chromatography. In general, all operations are carried out at 4 ° . The normal starting point for partial purification is an osmotic shockate or a whole-cell lysate S100. Streptomycin sulfate is added to the cold enzyme solution to a final concentration of 1.5%. After the streptomycin has dissolved, the mixture is mixed gently for 15 rain and then centrifuged at 15,000 g for 15 rain. The enzyme is precipitated from the supernatant by a suitable ammonium sulfate cut (this usually lies between 30 and 60% saturation with ammonium sulfate); the precipitate from the final cut is resuspended in and dialyzed against a suitable buffer and absorbed 13G. Cerny and M. Teuber, Arch. Mikrobiol. 78, 166 (1971). 1'O. Doi, S. Kondo, N. Tanaka, and H. Umezawa, J. Antibiot. 22, 273 (1969). ~Y. Sakagami, N. Takaishi, and A. Hachimori, J. Antibiot. 27, 248 (1974). ~"M. J. Haas, unpublished data. 17A. L. Smith and D. H. Smith, g. Inject. Dis. 129, 391 (1974). ~8M. Brzezinska and J. Davies, Antimicrob. Ag. Chemother. 3, 266 (1973).
[48]
AMINOGLYCOSIDE-M ODIFYING ENZYMES
603
to a DEAE-eellulose column. The column is washed with a small volume of buffer, and protein is eluted with buffer containing a linear sodium chloride, ammonium chloride, or potassium acetate concentration gradient from 0 to 0.5 M. Such a procedure usually results in a 25-70% increase in specific activity with a recovery of 3o-7o% of the initial activity.
Properties General Properties. The aminoglycoside-modifying enzymes are poorly characterized from a physical and chemical point of view. Most of the enzymes are inactivated by repeated freezing and thawing and are stable at --70 °. Substrate inhibition is commonly observed. The molecular weights of several of the enzymes have been estimated at 20,000-30,000. TM In general, a suitable buffer for dialysis, storage and assay contains 10 mM Tris-ehloride or acetate, 10 mM magnesium chloride or acetate, 25 mM ammonium chloride, and a sulfhydryl reagent (DTT, 0.5 raM-1 mM or fl-mercaptoenthanol, 1 mM-3 mM), pH 7.0-8.0. Kanamgcin AcetyltransJeq'ase (E. coli R5/~:6771';; NR79/W677 ~) Stability and Activity. In unbuffered osmotic shockates the enzyme loses 8% of its activity during 4.5 hr of incubation at 30 °. The presence of 5-25% glycerol increases stability but decreases activity by as much as 30%. The enzyme is stable at --70 ° and is stable to lyophilization and acetone precipitation. The pH optimmn for the acetylation of the kanamycins, neomycins, and tobramycin is approximately 5.8; the optimum for gentamicins C1 and C1,~is near 7.6. Enzyme stability and activity are reduced by concentrations of DTT above 5 mM. EC]ect o] BuJ~ers and Ions. The standard buffcr is buffer III. Tricine, MOPS, and cacodylate buffers at 10 mM are satisfactory. In phosphate, HEPES, Kolthoff-borate-phosphate, and Tris-maleate buffers the enzyme is less stable. The enzyme requires 10 mM magnesimn for activity but is inhibited by concentrations above 20 raM. The enzyme is unstable in sodium chloride but is stable in potassium acetate. Kinetic Data. The Kn, for the aeetylation of kanamyein A is 1 ~M at pH 7.0. The V..... is 1 ~mole/min using the assay conditions described. The acetylation of kanamyein is inhibited by paromomyein, paromamine, and gentamiein A. Substrate inhibition occurs at drug concentrations above 0.1 raM. ' ' J . Davfi~s and R. Benvcniste, Ann. N . Y . Acad. Sci. 235, 130 (1974).
624
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
Kanamycin Acetyltrans]erase (P. aeruginosa 37962°) Stability. KAT-3796 is stabilized by glycerol and ammonium chloride. The standard working buffer is buffer IV. Activity. The pH optimum for the acetylation of kanamycin A is between 5.6 and 6.5, and no pH optimum is exhibited between 5 and 9 for the acetylation of sisomicin and gentamicin C1~. The acetylation of kanamycin A, neomycin B and gentamicin Cla is inhibited by kanamycin C, gentamicin A, and, to a lesser extent, lividomycin.
Gentamicin Acetyltrans]erase I (E. coli R135/C6002°) Stability. The enzyme is stable at --20 ° and rapidly loses activity above 35 °. It is generally less stable than a similar enzyme isolated from P. aeruginosa 130. The standard buffer is buffer V. Kinetic Data. The Km for the acetylation of sisomicin is approximately 10 pjl//, and the V~ax is 0.9 nmole/min. The enzyme is inhibited by concentrations of sisomicin (but not of gentamicin C1) above 0.1 raM. Gentamicin Acetyltrans]erase I (P. aeruginosa 1302°) Stability. The enzyme is stable at --20 ° and is rapidly inactivated above 35 °. Buffer V is the standard buffer. pH Optimum. The pH optimum for the acetylation of sisomicin and kanamycin B is about 7.4. Gentamicin C1 acetylation exhibits a broad pH optimum (pH 5.4-8.0). Kinetic Data. The K,, for sisomicin acetylation is approximately 0.1 mM and the V~ax is about 60 nmole/min. The enzyme is inhibited by sisomicin, but not gentamicin C~, at concentrations above 0.1 mM. Gentamicin Acetyltrans]erase II (Providencia 16421) Stability. The enzyme is stable at --20 ° . Reactions are routinely supplemented with magnesium acetate and DTT although an absolute requirement for these has not been demonstrated. Activity. The pH optimum for acetylation of kanamycin C is 6.0; for gentamicin C1 it is 6.6. The enzyme is inhibited by poor substrates and nonsubstrates. 20S. Biddlecome, M. S. thesis, University of Wisconsin,Madison, 1973. 51M. Chevereau, P. J. L. Daniels, J. Davies, and F. LeGoffic, Biochemistry 13, 598 (1974).
[48]
AMI NOGLYCOSIDE-MODIFYING ENZYMES
625
Gentamicin Acetyltrans]erase III (P. aeruginosa PST-1 ~°) Stability. The enzyme is stable at --20 ° . It is not inactivated by incubation at 30 ° for 20 min. Activity. The pH optimum for the acetylation of the kanamycins, gentamicins, and neomycins is between pH 5.5 and 6.0. Magnesium (10 mM optimum) and a sulfhydryl reagent (DTT, 3 mM optimum) are required for activity but not for stability. Substrate inhibition occurs at varying concentrations with different substrates. Gentamicin A denylytrans]erase (E. coli JR66/W67722,-~:~) Requirements Jor Activity. DTT (1-10 mM) and magnesium are necessary for the maintenance of activity. Removal of magnesium results in irreversible inactivation. The enzyme is inhibited by high salt concentrations. pH Optimum. The pH optimum for the adenylylation of the gentamicins and kanamycins is 8.0-8.2. Streptomycin-Spectinomycin AdenylyltransJerase (E. coli NR73/W67724) Stability and Activation. The enzyme is stable at --10 ° and is activated by concentrations of ammonium chloride above 1 M. Ion and Co]actor Requirements. For the maintenance of activity and stability, 10 mM magnesium is required and may be replaced by manganese (0.1 mM), zinc, or cadmium. The enzyme will use ATP or dATP as cofactors. Kinetic Data. The Km's for the adenylylation of streptomycin and spectinomycin are 20 t~M. Activity is 50% reduced by 30 t~M pyrophosphate, but not by azide, iodide, cyanide, or pCMB. The enzyme is inhibited by streptomycin or spectinomycin at 0.2 mM. Streptomycin adenylylation is inhibited by tetracycline. Streptomycin-Spectinomycin AdenylyltransJe,'ase (E. coli B/RE130 '-'~) Activity Requirements. The enzyme requires magnesium (8-10 mM) and a reducing agent for maximal activity. Magnesium cannot be re52R. Benveniste, Ph.D. thesis, University of Wisconsin, Madison, 1972. 53M. Yagisawa, H. Naganawa, S. Kondo, M. Hamada, T. Takeuchi, H. Umezawa, J. Antibiot. 24, 911 (1971). 2~R. Benveniste, M. S. thesis, University of Wisconsin, Madison, 1970. 25j. H. Harwood and D. H. Smith, J. Bacteriol. 97, 1262 (1969).
626
ANTIBIOTIC INACTIVATION AND MODIFICATION
[48]
placed by nickel, cobalt, zinc, calcium, or manganese. The pH optimum for the adenylylation of streptomycin is near 8.3. The enzyme will use ATP and dATP as cofactors.
Neomycin Phosphotrans]erase I (E. coli JR35/W677:6,27; JR39/W6772s; ML162929)
Activity and Stability. The enzyme from JR39 has an absolute requirement for magnesium and is stabilized by 0.1 M ammonium chloride or 10 ~g/ml neomycin B. The enzyme from ML1629 is inactivated by 5 min of incubation at 45 °. It also requires magnesium. DTT, 10 mM, prevents denaturation and restores the activity of denatured enzyme. Inhibition. Phosphorylation of neomycin B is inhibited by gentamicin C1~ and by tobramycin. Neomycin Phosphotrans]erase H (E. coli JR66/W677~s,29; P. aeruginosa Ps49 ~8) Stability. The enzyme is stabilized by 10 ~g/ml neomycin B or 0.1 M ammonium chloride. Activity. There is no marked pH optimum between pH 5.5 and 8.0 for the phosphorylation of neomycin B. Neomycin B phosphorylation is not inhibited by tobramycin or by gentamicin C1~ but phosphorylation of kanamycin C is inhibited by tobramycin. Neomycin PhosphotransJerase (P. aeruginosa H-93°) Stability. The phosphorylation of kanamycin proceeds for 20 rain at 55 °, for 5 min at 65 ° and not at all at 75% pH and heat stability are not markedly affected by 10 mM EDTA, 10 mM magnesium acetate, or 0.4 mM kanamycin. The enzyme is stable between pH 7.5 and 9. pH and Kinetic Data. The pH optimum for the phosphorylation of kanamycin is 7.5. At this pH, the Km for the phosphorylation of kanamycin is 0.3 raM, and the Vm~xis 42 ~moles/hr/OD unit. Ion and Cofactor Requirements. The enzyme requires magnesium, manganese, zinc, or cobalt (all at 1 raM) for activity. ATP is the only ~ M. S. Okanishi, S. Kondo, R. Utahara, and H. Umezawa, J. Antibiot. 21, 13 (1967). 21B. Ozanne, R. Benveniste, D. Tipper, and J. Davies, J. Bacteriol. 100, 1144 (1969). M. Brzezinska and J. Davies, Antimicrob. Ag. Chemother. 3, 266 (1973). 20M. Yagisawa, H. Yamamoto, I-I. Naganawa, S. Kondo, T. Takeuchi, and H. Umezawa, J. Antibiot. 25, 748 (1972). 3, O. Doi, S. Kondo, N. Tanaka, and H. Umezawa, J. Antibiot. 22, 273 (1969).
[48]
AMINOGLYCOSIDE-MODIFYING ENZYMES
627
accepted phosphoryl donor when partially purified enzyme preparations are used; crude preparations will use other nucleoside triphosphates. One hundred percent of the available kanamycin is modified in the presence of equimolar ATP.
Neomycin Phosphotrans/erase (S. aureus B294 :~) pH Optimum. The pH optimum for the inactivation of kanamycin is between pH 7.0 and 7.5. Inhibition. Substrate inhibition occurs at a kanamycin concentration of 2 m M but not at 0.2 mM. Lividomycin Phosphotrans/erase (E. coli R,,~+/ML14103'-'; P. cleruginosa Ti-1333; S. aureus KW-234)
Activity Requirements. The three enzymes require A T P and magnesium for activity.
pH Optimum. The pH optimum for the phosphorylation of lividomycin is between 6.5 and 7.0 for the three enzymes. Streptomycin Phosphotrans/erase (E. coli JR35/W677 '-,T) Ion and Co/actor Requirements. Magnesium is required for maximal activity, but not for stability, and can be partially replaced by zinc or manganese but not by copper (all at 5 raM). The enzyme uses A T P or G T P (but not C T P or U T P ) . pH Optimum. The pH optimum for the phosphorylation of strei)tomycin is 8.0. Streptomycin Phosphotransferase (P. aeruginosa H-93~) pH Optimum. The pH optimum for the phosphorylation of streptomycin is 8.5.
Co/actor Requirements. The enzyme will use ATP, CTP, GTP, or U T P ~' O. Doi, M. Miyamoto, N. Tanaka, and H. Vmezawa, Appl. Microbiol. 16, 1282 (1968). 3;M. Yamaguchi, T. Koshi, F. Kobayashi, and S. Mitsuhashi, Antin~icrob. Ag. Chemother. 2, 145 (1972). ~F. Kobayashi, M. Yarnaguchi, and S. Mitsuhashi, Antimic~'ob. Ag. Chemother. 1, 17 (1972). ~ F. Kobayashi, T. Koshi, J. Eda, Y. Yoshimura, and S. Mitsuhashi, Antimicrob. Ag. Chemother. 4, 1 (1973). '~ O. Doi, M. Ogura, N. Tanaka, and H. Umezawa, Appl. Microbiol. 16, 1276 (1968).
628
[49]
ANTIBIOTIC INACTIVATION AND MODIFICATION
as cofactors; maximal activity is observed at an ATP concentration of 2O mM. Acknowledgment We thank Dr. Julian Davies for many useful contributions to this work.
[49] ATP:Streptomycin 6-Phosphotransferase: By JAMES B. WALKER Dihydrostreptomycin +
MgATP
'l
NH,
MgADP
L~---o ,\
.,c~/, OH
C:NH + I NH
+
OH
I
J - - - - t ~,=H~
, "Kc.,o. I
NH z
/,'-O.~oH
,
(1)
)~
oPO;-
I
Dihydrostreptomycin-6-P
H~N--C--NH
Streptidine
+
MgATP
,
NH2 I + C=NH,
~ MgADP
(2) tt
oPofSt reptidine - 6 - P
Streptomycin 6-kinase is present in high concentration in extracts of streptomycin-producing strains of Streptomyces. 2-~ This enzyme can also 1EC 2.7.1.72 (mistakenly called 5-kinase). "J. B. Walker and M. S. Walker, Biochim. Biophys. Acta 148, 335 (1967). 3 A. L. Miller and J. B. Walker, J. BacteTiol. 99, 401 (1969). , M. S. Walker and J. B. Walker, J. Biol. Chem. 245, 6683 (1970). O. Nimi, G. Ito, Y. Ohata, S. Funayama, and R. Nomi, Agr. Biol. Chem. 3~, 850 (1971).
