Expression, Purification and Crystallization Vibrio harveyi Acyltransferase
of the
Lora Swenson’, Stefano R. Ferri’, Ruth Green’, Allan M. Sharp’ Edward A. Meighen2 and Zygmunt S. Derewenda’? ‘Medical
Research Council of Canada Group in Protein Structure and Functio,n Department of Biochemistry, University of Alberta, Edmonton, Alberta,, T6G 2H7, Canada ‘Department of Biochemistry, McGill University Montreal, Quebec, H3G 7 Y6. C:anada (Received 23 March
1992; accepted 1S May
1992)
We have obtained X-ray quality single crystals of Vibrio harveyi acyltransferase. The protein was obtained from 1’. harveyi by a gene mobilization expression system. The crystals are monoclinic (space group P2,. a = 89.9 8, h = 83% 8, c = 47.1 A. fl = 97.3”) w&h two molecules related by a pronounced non-crystallographic dyad in the asymmetric unit. with a solvent content of approximately riO”/b. The diffraction pattern from fresh crystals extends beyond 2 A resolution using sealed tube @uKa radiation. The elucidation of the three-dimensional structure of this enzyme, believed to contain a proteinase-like catalytic, triad, which resembles in many ways other eukaryotic fatty acid chain terminat,ing enzymes, may have important consequences for our understanding of the molecular basis of the final stages of the synthesis of fatty acids. Keywords:
protein
crystallization:
Tn both prokaryotes and eukaryotes. fatty acid synthesis terminates with the hydrolysis or transfer of the growing acyl chain located on a 4’-phosphopantetheine prosthetic group (Rock & Cronan. 198.5: Wakil et al., 1983). The fate of this acyl group depends upon the species of the organism. For example, in Escherichia CC&, acyl chains reach a length of about 16 carbons, at which point they are transferred directly into lipids by a family of specific transferases, acyltransferases. Tn yeast and the phylogenetically advanced bacterium, Mycobacterium smegmatis, a similar function is carried out by an acyltransferase domain of the fatty acid synthase, which transfers the acyl chain to coenzyme A (CoA). Tn animal cells, a thioesterase domain of the fatty acid synt’hase hydrolyzes t,he growing acyl chain when it reaches the appropriat,e length, i.e. that of a palmitic acid. In some specific3 vertebrate tissues, such as the mammary gland of non-ruminant mammals (Knudsen it al.. 1976: Libertini et al., 1976) and the uropygial gland of certain waterfowl (de Renobales et al.. 198(I), a different enzyme, thioesterase Tl, is responsible for t Author to whom all correspondence should lw addressed.
acyltransferase
the release of medium chain length fatty acids. To date there is no st,ruct’ural information with respect to any of these enzymes. In light emitting ba&eria, a 1jrx-specific8 acoy]transferase, with a similar chain terminating funcation. is responsible for specifically diverting myristico acid from fatty acid biosynthesis to the luminescent system (Ferri & Meighen. 1991). The enzyme is also capable of transferring the acyl group from acylACP and acyl-(loA to glycerol, ethylene gly~ol and fl-mercaptoethanol (Carey et al., 1984). This acylt,ransferase is part of a fatty acid reductase complex. which includes acyl-protein synthase and acyl-(.‘()A f’or responsible reducta.sc subunits t tw ATP-dependent activat,ion and hTAI)PH-tlrl)~.tldent respectivrly. of t hp myrist ic* ;t(.id reduction. provided by the ac*yltransferase (Meighetl. 1!,!,I). The resulting aldehpde is then oxidized to thr eorrv’sponding fatty acid by Iuciferase in a FMNH,anal (),-dependent reaction. accompanied by the tmission of light. Although a fatty acid is thr product of’ this react>ion, a caonstant supply has to bt> maintained by t’he acyltransferase, possibly due to the, preseme of a competing acyl-=\(‘I’ synt)ha,scx. which ligates free fatty acids t.o acyl carrier proteins (Ryers & Holmes, 1990).
Crystallization
Most fatty acid chain terminating enzymes appear to he serine hydrolases. Their active nucleophilic serine is contained within a pentapeptide 0;.X,-S-X,-C common to both e&erases and neutral lipases (Rrenner, 1988). Recent crystallographic investigations of lipases from Rhizomucor miehei (Brady et al.? 1990), human pancreas (Winkler et al., 1990) and Beotrichum candidum (Schrag et al., 1991) and of Torpedo californica acetylcholine esterase (Sussman et al., 1991) revealed that in each of the cases studied so far the active serine is a part of a hydrogen-bonded t’riad including a histidine and Asp/Ulu. thus making the active centers very reminiscent of t,he serine proteinases (Blow et al.: 1969). The lux-specific acyltransferase is also a serine est,erasr as shown by the presence of the C-X,-S-X,-G motif, inactivation with phenylmethanesulfonyl fluoride, stability of the acylatrd enzyme intermediate to cleavage by neutral hydroxylamine, and site-directed mutagenesis (Ferri & Meighen, 1991). The dependence of t,he enzyme activit’y on a basic group with a ph’, of 6.3 and similarities in the amino acid sequence with other t,hioesterases also suggest the presence of an essent’ial hist,idine residue as part of a catalytic triad. ln this regard, an essential histidine residue in the rat mammary bhioesterase TI has recently been identitied (Witkowski et al., 1991). In this communication w-e report’ the expression and purification of the 1,‘. harwyi acyltransferase and the preparation of X-ray quality single cryst’als. The study was underinitiate detailed examination of taken to st.ructure--fun&ion relationships in this, hitherto struc>t,urally uncaharact’erized, family of enzymes. A 1600 base-pair IIFlpaII restriction fragment containing thfb entire ZusD gene encoding the acyltransferase of 1’. harveyi (R392) was inserted downstream frotn a 1’. hnrvpyi-induced promoter located in the cloning ve&or pMGM1 10. derived from pKT230 (Miyamot’o et (IL., 1990; Ferri et nt decrease in acpltrans-
Notes
573
ferasr activity. The precipitate was then resuspended in 100 ml 50 mu-R;a,/K phosphate (pH (buffer A) and 7.0), 10 mM-/?-mercaptoethanol dialyzed overnight. The dialyzed fraction was applied to a DEAE-Sepharose column (2.5 cm x 20 cm). washed with 100 ml buff’er A, and eluted with a linear 0 M to 0.5 M-NaCI gradient, (400 ml in total) in buffer A. The pooled fractions were precipitated wit,h 75 y0 saturated ammonium sulfate to form two pellets. Each was dissolved in % ml buffer A and applied on an Cltrogel AcA54 gel tilt’ration column (1.5 cm x 48 cm) and eluted with buffer A uit,h a flow rate of 20 ml/h. The pooled fractions (in i,‘OO,Aglycerol) were st,ored at - 20°C. The protein appeared as a single band on tiJN/I’AGE and the previously used hydroxyapatite chromat,ography step was not, necessary. Single crystals of the V. hareeyi acylt’ransferase were grown using the hanging drop method: 3 ~1 of the protein solution (9 mg/ml, 100 mM-potassium phosphate buffer. pH 7.0. 200;, glFcero1) were added to 3 ~1 of thr reservoir solution (550;;, saturated ammonium sulfate. 100 mM-potassium phosphate buffer, pH 6.0, I?;, polyethylene glycol iVr. 200) and suspended over the reservoir. Crystals appeared after about one week and took several weeks to reach their maximum size. A small crvstal (0.3 mm x 0.1 mm x0.2 mm) was used for &itial X-ray dat,a collection with a Sirmens (Xentronics) area detector. mount,ed on a conventional sealedtube source with a Vu target and a ivi filter. The dct,ertor was placed 15 cm away frorn the sample: 0.25” images were processed using the XENGEN (Howard et nl.. 1987) software pa,ckage and the unit) wII was identified aut,omatioally as monoclinic ((I = 89.9 A. h = 83.6 13. c = 47.1 Ii. B = 97.3”: 1 ,A = &l nm). The st,andard crystallographic merging R factor for this data set. which extended to 4.4 ‘4 resolution, was 0.063. Weighted reciprocal lattice diagrams were plotted using the I,CFPR,EC program ((VP4 cxrystallographic software package. SERC’ Daresbury Laborat,ory. C’.K.) and no additional symmet,ry elements were found. In addition, svst’ernatic absences were found to occur along the (I”* axis (/c = 2n). It was therefore concluded that the unit cell was indeed monoclinic and thr space group was assigned as 1’2,. Taking into account the calcu1at)c.d molecular weight of 34.168 daltons for the 1’. harwyi acytt,ransferasr. two molecules per asymmetric unit, result in a specifics volumr value of 2.,% A3il)a. Since this pointed t’o a dimer in t’he asymmetric. unit we decided t,o investipatc> the possibility of non-crystallographic symmet,r\-. A large single crystal was used this timca to collect high resolution data using the same Siemens area de&&or system, except that the chamber was posit,iotred 11 cm from the sample. At the beginning the crystal diffract,rd to -2.0 ,4, but radiation damage observed during the course of t.h(x experiment decreased the effective resolution to - 2.4 -4: 88,173 observations were reduced to 30.517 unique reflections with a merging R factor of W82. These dat,a wt‘rc used t>o c~alculat,r a self rotation fun&on (in
spherical polar co-ordinates) using the program I’OLARSFRF from the WY4 suit,e. X single peak, with the magnitude of 95(“,, of the origin peak was found on t,he ti = 180” section for C#J = 6+-S” and w = 90”. This non-c*rystallographic symmetry (~1 be clrarly observed on the hOZ zone of X-ray dat,a. A high value of the self-rotation function maximum is usually observed when the protein is highly ordered and contains a significant percentagcb of well-defined secondary structure elements. A search for heavy-atom derivatives is under
mallartl
ducks
J~iophy.~.
205.
(Anas 1,latyrh~ytLcho.s). 1W li7.
.-I rch.
f-Goch,rm
Petri. S. B ~llrighrll. E. (19!11). A lu.j:-specific myGstoyl to in luminescent bacbtrria related transferase eukaryotic, serin( &erases. ./. /Zio/. (‘h~tu. 266, I2852 12857. Ferri. S.. Soly. It.. Miyarnot’o. c’. &, Mrighen. E. (1991). (‘omplt,merltation in U~VOof the Zux-speczific fatty acid reductasr subunits from different, luminrscent bacateria. III WiolurrLinescrrcc~~ and (‘h~trliluntittr.sc~ttc~. C‘urr~n! Stabs (Stanley. I’. & Kricka. I,.. cds). pp, 39- 4%. .I. \C’ilry & Sons, England. Howard. A. -1.. (~illilancl. G. I,., Finzel, B. C’.. l’oulous. T. TA..Ohlendorf, I). H. B Sallemmr, F. (1987). I’SV of an imaging proportional rounter in niacromole~~ular 20, 38:j -M7. c+rystallography. J. A&. (‘rysfnlloyr. Knudsen. .I.. Clark. S. Jt Dils. R. (1976). Purification and some properties ot ii medium-c*hain acay-thiotsster hydrolase from lactating-rabbit mammary gland which trrminat,es c.hain elongation in fat,ty aceid synthesis. Hiochem. .J. 160. 683-691. Libertini. I,.. I,in. (‘. & Smith. H. (1976). Isolation and proprrtirs of two different thioest.erasrs from rat t,issues. li’rri. l’roc. Fud. ;Itttrr. &or. lG:.rp. Hiol. 35. 1671. Meighen. E. .A. (1991). Molecular biology of bac*trrial bioluminescence. ;MicroDiol. Met>.55. 1X-142. Miyam&o. 1’.. Meighen. E. & Graham. A. (1990). Transcriptional regulation of Zlr.r genes transfcarred 172. 2046--‘LOf,S. into L’ihrio hnrcqi. .J. Racturiol. Rock. (‘. cir Cronan. *J. (1985). Lipid metabolism in procaryotes. In HiochwListry qf Lipids rrtrd Membrane.r; (Vance. I). 8z Vance. J.. eds), pp 73 Il.‘. Benhamin-(“ummings. Menlo Park. (:A. Schrag, J. I).. Li. Y.. Wu, S. $ (1ygler. M. (1991). Srr-His-(:lu forms the catalytic site of a lipase from I 351. geotrichurn candidum. .%akclrr (London)
Wag.
This study was financed hy the Medical Research Council of Canada grant to the Croup in Protein and Function (Z.S.D.) and to E.A.M Structure (MT-4314). We thank Dr I’. Derewenda for help with comput,ing, Dr Y. Wri for help with data &lection and Koto Hayakawa for help with growing crystals.
References Blow. I). M., Birktoft. J. & Hartley, B. (1969). Role of a buried acid group in the mechanism of action of rhymotrypsin. Nature (London), 221, 33-339. Brady. I,.. Brzozowski. A.. Derewenda. Z., Dodson. E., Dodson, G., Tolley, S.. Turkenburg, J.. Christiansen. I,.. Huge-Jensen, B.. Norskow. I,., Thim. L. & Menge. IT. (1990). A serine protease triad forms the catalytic (London). centre of a triacglglycerol lipasr. Xatwe 343. 767-770. Brenner, S. (1988). The molecular evolution of genes and proteins: A tale of two serines. .~alure (London). 334. 528-530.
Scil-i6l.
Byrrs, I). & Holmes, (1. (1990). A soluble fatty acByl-acyl carrier protein synthetase from the bioluminescent harveyi. Biochrm. Cell Biol. 68. bac%erium Vibrio 1045- 1051. Byrrs, I). & Meighhen, E. (1985). Purification and characterization of a bioluminescence-related fatty acyl esterase from Vibrio harvey. J. Viol. (‘hem. 260.
Sussman. *J. I,.. Harrl. M., Frolow. F., Orfner, C’., Goldman, A., Toker. I,. b; Silman. I. (1991). ?\tornic* structure of acetylcholinesterase from Torpedo cnlifbrnica: A prototypic aretylcholine-hind protein. Scirncr.
6938 - 6944.
C’arry, L.. Rodringuez. A. & Meighen, E. ( 1981). Generation of fatty acids by an acyl esterase in the of Photobacterium system bioluminescent phosphoreum. ,J. Biol. Chem. 259. 10216-10221. deRenobales, M., Rogers, 1,. & Kolattukudy. P. (1980). Involvement of a thioesterase in the production of short-chain fatt,y acids in t.he uropygial glands of
Edited
by
253.
872-879.
Wakil. S.. Stoops. J. & %Joshi. V. (1983). Fatty acid synthesis and its regulat,ion. dnnw Rw. Hiochem. 52, 5377579. Winkler, F. K.. D’Arcy, ;1. &. Hunziker, W. (1990). Structure of human pancreatic lipasr. .Vrctwe (London),
343.
771-774.
Witkowski. A.. Naggert, J.. Wessa, B. & Smith. S. (1991). A catalytic role for histidine 237 in rat mammary gland thioesterase TI. .I. Hiol. (‘hem. 266. 18514 18519.
K.
Huber
of the
Lora Swenson’, Stefano R. Ferri’, Ruth Green’, Allan M. Sharp’ Edward A. Meighen2 and Zygmunt S. Derewenda’? ‘Medical
Research Council of Canada Group in Protein Structure and Functio,n Department of Biochemistry, University of Alberta, Edmonton, Alberta,, T6G 2H7, Canada ‘Department of Biochemistry, McGill University Montreal, Quebec, H3G 7 Y6. C:anada (Received 23 March
1992; accepted 1S May
1992)
We have obtained X-ray quality single crystals of Vibrio harveyi acyltransferase. The protein was obtained from 1’. harveyi by a gene mobilization expression system. The crystals are monoclinic (space group P2,. a = 89.9 8, h = 83% 8, c = 47.1 A. fl = 97.3”) w&h two molecules related by a pronounced non-crystallographic dyad in the asymmetric unit. with a solvent content of approximately riO”/b. The diffraction pattern from fresh crystals extends beyond 2 A resolution using sealed tube @uKa radiation. The elucidation of the three-dimensional structure of this enzyme, believed to contain a proteinase-like catalytic, triad, which resembles in many ways other eukaryotic fatty acid chain terminat,ing enzymes, may have important consequences for our understanding of the molecular basis of the final stages of the synthesis of fatty acids. Keywords:
protein
crystallization:
Tn both prokaryotes and eukaryotes. fatty acid synthesis terminates with the hydrolysis or transfer of the growing acyl chain located on a 4’-phosphopantetheine prosthetic group (Rock & Cronan. 198.5: Wakil et al., 1983). The fate of this acyl group depends upon the species of the organism. For example, in Escherichia CC&, acyl chains reach a length of about 16 carbons, at which point they are transferred directly into lipids by a family of specific transferases, acyltransferases. Tn yeast and the phylogenetically advanced bacterium, Mycobacterium smegmatis, a similar function is carried out by an acyltransferase domain of the fatty acid synthase, which transfers the acyl chain to coenzyme A (CoA). Tn animal cells, a thioesterase domain of the fatty acid synt’hase hydrolyzes t,he growing acyl chain when it reaches the appropriat,e length, i.e. that of a palmitic acid. In some specific3 vertebrate tissues, such as the mammary gland of non-ruminant mammals (Knudsen it al.. 1976: Libertini et al., 1976) and the uropygial gland of certain waterfowl (de Renobales et al.. 198(I), a different enzyme, thioesterase Tl, is responsible for t Author to whom all correspondence should lw addressed.
acyltransferase
the release of medium chain length fatty acids. To date there is no st,ruct’ural information with respect to any of these enzymes. In light emitting ba&eria, a 1jrx-specific8 acoy]transferase, with a similar chain terminating funcation. is responsible for specifically diverting myristico acid from fatty acid biosynthesis to the luminescent system (Ferri & Meighen. 1991). The enzyme is also capable of transferring the acyl group from acylACP and acyl-(loA to glycerol, ethylene gly~ol and fl-mercaptoethanol (Carey et al., 1984). This acylt,ransferase is part of a fatty acid reductase complex. which includes acyl-protein synthase and acyl-(.‘()A f’or responsible reducta.sc subunits t tw ATP-dependent activat,ion and hTAI)PH-tlrl)~.tldent respectivrly. of t hp myrist ic* ;t(.id reduction. provided by the ac*yltransferase (Meighetl. 1!,!,I). The resulting aldehpde is then oxidized to thr eorrv’sponding fatty acid by Iuciferase in a FMNH,anal (),-dependent reaction. accompanied by the tmission of light. Although a fatty acid is thr product of’ this react>ion, a caonstant supply has to bt> maintained by t’he acyltransferase, possibly due to the, preseme of a competing acyl-=\(‘I’ synt)ha,scx. which ligates free fatty acids t.o acyl carrier proteins (Ryers & Holmes, 1990).
Crystallization
Most fatty acid chain terminating enzymes appear to he serine hydrolases. Their active nucleophilic serine is contained within a pentapeptide 0;.X,-S-X,-C common to both e&erases and neutral lipases (Rrenner, 1988). Recent crystallographic investigations of lipases from Rhizomucor miehei (Brady et al.? 1990), human pancreas (Winkler et al., 1990) and Beotrichum candidum (Schrag et al., 1991) and of Torpedo californica acetylcholine esterase (Sussman et al., 1991) revealed that in each of the cases studied so far the active serine is a part of a hydrogen-bonded t’riad including a histidine and Asp/Ulu. thus making the active centers very reminiscent of t,he serine proteinases (Blow et al.: 1969). The lux-specific acyltransferase is also a serine est,erasr as shown by the presence of the C-X,-S-X,-G motif, inactivation with phenylmethanesulfonyl fluoride, stability of the acylatrd enzyme intermediate to cleavage by neutral hydroxylamine, and site-directed mutagenesis (Ferri & Meighen, 1991). The dependence of t,he enzyme activit’y on a basic group with a ph’, of 6.3 and similarities in the amino acid sequence with other t,hioesterases also suggest the presence of an essent’ial hist,idine residue as part of a catalytic triad. ln this regard, an essential histidine residue in the rat mammary bhioesterase TI has recently been identitied (Witkowski et al., 1991). In this communication w-e report’ the expression and purification of the 1,‘. harwyi acyltransferase and the preparation of X-ray quality single cryst’als. The study was underinitiate detailed examination of taken to st.ructure--fun&ion relationships in this, hitherto struc>t,urally uncaharact’erized, family of enzymes. A 1600 base-pair IIFlpaII restriction fragment containing thfb entire ZusD gene encoding the acyltransferase of 1’. harveyi (R392) was inserted downstream frotn a 1’. hnrvpyi-induced promoter located in the cloning ve&or pMGM1 10. derived from pKT230 (Miyamot’o et (IL., 1990; Ferri et nt decrease in acpltrans-
Notes
573
ferasr activity. The precipitate was then resuspended in 100 ml 50 mu-R;a,/K phosphate (pH (buffer A) and 7.0), 10 mM-/?-mercaptoethanol dialyzed overnight. The dialyzed fraction was applied to a DEAE-Sepharose column (2.5 cm x 20 cm). washed with 100 ml buff’er A, and eluted with a linear 0 M to 0.5 M-NaCI gradient, (400 ml in total) in buffer A. The pooled fractions were precipitated wit,h 75 y0 saturated ammonium sulfate to form two pellets. Each was dissolved in % ml buffer A and applied on an Cltrogel AcA54 gel tilt’ration column (1.5 cm x 48 cm) and eluted with buffer A uit,h a flow rate of 20 ml/h. The pooled fractions (in i,‘OO,Aglycerol) were st,ored at - 20°C. The protein appeared as a single band on tiJN/I’AGE and the previously used hydroxyapatite chromat,ography step was not, necessary. Single crystals of the V. hareeyi acylt’ransferase were grown using the hanging drop method: 3 ~1 of the protein solution (9 mg/ml, 100 mM-potassium phosphate buffer. pH 7.0. 200;, glFcero1) were added to 3 ~1 of thr reservoir solution (550;;, saturated ammonium sulfate. 100 mM-potassium phosphate buffer, pH 6.0, I?;, polyethylene glycol iVr. 200) and suspended over the reservoir. Crystals appeared after about one week and took several weeks to reach their maximum size. A small crvstal (0.3 mm x 0.1 mm x0.2 mm) was used for &itial X-ray dat,a collection with a Sirmens (Xentronics) area detector. mount,ed on a conventional sealedtube source with a Vu target and a ivi filter. The dct,ertor was placed 15 cm away frorn the sample: 0.25” images were processed using the XENGEN (Howard et nl.. 1987) software pa,ckage and the unit) wII was identified aut,omatioally as monoclinic ((I = 89.9 A. h = 83.6 13. c = 47.1 Ii. B = 97.3”: 1 ,A = &l nm). The st,andard crystallographic merging R factor for this data set. which extended to 4.4 ‘4 resolution, was 0.063. Weighted reciprocal lattice diagrams were plotted using the I,CFPR,EC program ((VP4 cxrystallographic software package. SERC’ Daresbury Laborat,ory. C’.K.) and no additional symmet,ry elements were found. In addition, svst’ernatic absences were found to occur along the (I”* axis (/c = 2n). It was therefore concluded that the unit cell was indeed monoclinic and thr space group was assigned as 1’2,. Taking into account the calcu1at)c.d molecular weight of 34.168 daltons for the 1’. harwyi acytt,ransferasr. two molecules per asymmetric unit, result in a specifics volumr value of 2.,% A3il)a. Since this pointed t’o a dimer in t’he asymmetric. unit we decided t,o investipatc> the possibility of non-crystallographic symmet,r\-. A large single crystal was used this timca to collect high resolution data using the same Siemens area de&&or system, except that the chamber was posit,iotred 11 cm from the sample. At the beginning the crystal diffract,rd to -2.0 ,4, but radiation damage observed during the course of t.h(x experiment decreased the effective resolution to - 2.4 -4: 88,173 observations were reduced to 30.517 unique reflections with a merging R factor of W82. These dat,a wt‘rc used t>o c~alculat,r a self rotation fun&on (in
spherical polar co-ordinates) using the program I’OLARSFRF from the WY4 suit,e. X single peak, with the magnitude of 95(“,, of the origin peak was found on t,he ti = 180” section for C#J = 6+-S” and w = 90”. This non-c*rystallographic symmetry (~1 be clrarly observed on the hOZ zone of X-ray dat,a. A high value of the self-rotation function maximum is usually observed when the protein is highly ordered and contains a significant percentagcb of well-defined secondary structure elements. A search for heavy-atom derivatives is under
mallartl
ducks
J~iophy.~.
205.
(Anas 1,latyrh~ytLcho.s). 1W li7.
.-I rch.
f-Goch,rm
Petri. S. B ~llrighrll. E. (19!11). A lu.j:-specific myGstoyl to in luminescent bacbtrria related transferase eukaryotic, serin( &erases. ./. /Zio/. (‘h~tu. 266, I2852 12857. Ferri. S.. Soly. It.. Miyarnot’o. c’. &, Mrighen. E. (1991). (‘omplt,merltation in U~VOof the Zux-speczific fatty acid reductasr subunits from different, luminrscent bacateria. III WiolurrLinescrrcc~~ and (‘h~trliluntittr.sc~ttc~. C‘urr~n! Stabs (Stanley. I’. & Kricka. I,.. cds). pp, 39- 4%. .I. \C’ilry & Sons, England. Howard. A. -1.. (~illilancl. G. I,., Finzel, B. C’.. l’oulous. T. TA..Ohlendorf, I). H. B Sallemmr, F. (1987). I’SV of an imaging proportional rounter in niacromole~~ular 20, 38:j -M7. c+rystallography. J. A&. (‘rysfnlloyr. Knudsen. .I.. Clark. S. Jt Dils. R. (1976). Purification and some properties ot ii medium-c*hain acay-thiotsster hydrolase from lactating-rabbit mammary gland which trrminat,es c.hain elongation in fat,ty aceid synthesis. Hiochem. .J. 160. 683-691. Libertini. I,.. I,in. (‘. & Smith. H. (1976). Isolation and proprrtirs of two different thioest.erasrs from rat t,issues. li’rri. l’roc. Fud. ;Itttrr. &or. lG:.rp. Hiol. 35. 1671. Meighen. E. .A. (1991). Molecular biology of bac*trrial bioluminescence. ;MicroDiol. Met>.55. 1X-142. Miyam&o. 1’.. Meighen. E. & Graham. A. (1990). Transcriptional regulation of Zlr.r genes transfcarred 172. 2046--‘LOf,S. into L’ihrio hnrcqi. .J. Racturiol. Rock. (‘. cir Cronan. *J. (1985). Lipid metabolism in procaryotes. In HiochwListry qf Lipids rrtrd Membrane.r; (Vance. I). 8z Vance. J.. eds), pp 73 Il.‘. Benhamin-(“ummings. Menlo Park. (:A. Schrag, J. I).. Li. Y.. Wu, S. $ (1ygler. M. (1991). Srr-His-(:lu forms the catalytic site of a lipase from I 351. geotrichurn candidum. .%akclrr (London)
Wag.
This study was financed hy the Medical Research Council of Canada grant to the Croup in Protein and Function (Z.S.D.) and to E.A.M Structure (MT-4314). We thank Dr I’. Derewenda for help with comput,ing, Dr Y. Wri for help with data &lection and Koto Hayakawa for help with growing crystals.
References Blow. I). M., Birktoft. J. & Hartley, B. (1969). Role of a buried acid group in the mechanism of action of rhymotrypsin. Nature (London), 221, 33-339. Brady. I,.. Brzozowski. A.. Derewenda. Z., Dodson. E., Dodson, G., Tolley, S.. Turkenburg, J.. Christiansen. I,.. Huge-Jensen, B.. Norskow. I,., Thim. L. & Menge. IT. (1990). A serine protease triad forms the catalytic (London). centre of a triacglglycerol lipasr. Xatwe 343. 767-770. Brenner, S. (1988). The molecular evolution of genes and proteins: A tale of two serines. .~alure (London). 334. 528-530.
Scil-i6l.
Byrrs, I). & Holmes, (1. (1990). A soluble fatty acByl-acyl carrier protein synthetase from the bioluminescent harveyi. Biochrm. Cell Biol. 68. bac%erium Vibrio 1045- 1051. Byrrs, I). & Meighhen, E. (1985). Purification and characterization of a bioluminescence-related fatty acyl esterase from Vibrio harvey. J. Viol. (‘hem. 260.
Sussman. *J. I,.. Harrl. M., Frolow. F., Orfner, C’., Goldman, A., Toker. I,. b; Silman. I. (1991). ?\tornic* structure of acetylcholinesterase from Torpedo cnlifbrnica: A prototypic aretylcholine-hind protein. Scirncr.
6938 - 6944.
C’arry, L.. Rodringuez. A. & Meighen, E. ( 1981). Generation of fatty acids by an acyl esterase in the of Photobacterium system bioluminescent phosphoreum. ,J. Biol. Chem. 259. 10216-10221. deRenobales, M., Rogers, 1,. & Kolattukudy. P. (1980). Involvement of a thioesterase in the production of short-chain fatt,y acids in t.he uropygial glands of
Edited
by
253.
872-879.
Wakil. S.. Stoops. J. & %Joshi. V. (1983). Fatty acid synthesis and its regulat,ion. dnnw Rw. Hiochem. 52, 5377579. Winkler, F. K.. D’Arcy, ;1. &. Hunziker, W. (1990). Structure of human pancreatic lipasr. .Vrctwe (London),
343.
771-774.
Witkowski. A.. Naggert, J.. Wessa, B. & Smith. S. (1991). A catalytic role for histidine 237 in rat mammary gland thioesterase TI. .I. Hiol. (‘hem. 266. 18514 18519.
K.
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