Vitamin B, and Decarboxylation of Histidine ESMOND E. SNELL Departments of Microbiology and Chemistry The University of Texas Austin, Texas 78712
In recent years a principal interest of my laboratory has been the amino acid decarboxylases, especially histidine decarboxylases. As it happens, development of knowledge of vitamin B, and of decarboxylases are intimately connected, and I therefore will present a brief retrospective view of this relationship before discussing some of our recent work on histidine decarboxylases.
VITAMIN B,, DECARBOXYLASES, AND PYRIDOXAL 5’ PHOSPHATE The confused early attempts at defining the nature of the B vitamins might be likened to solving a jigsaw puzzle from a bin full of mixed parts with no knowledge of the shape, size, or number of the pieces. The initial pieces are most difficult to identify, and the job becomes easier as more pieces are characterized. For nutritional biochemists, the initial task was to devise a diet from crude materials sufficiently low in a single trace substance so that impairment of growth (or some other observable function) of the test organism occurred that was prevented or corrected by supplementation with sources of the missing substance. Such an assay method for vitamin B, was achieved with rats in about 1934 (TABLE1) and made possible isolation of an active compound in five different laboratories in 1938. This accomplishment was followed by proof of structure and synthesis of pyridoxine in 1939. Pyridoxine and vitamin B, were then considered identical until 1942, when irregularities in the response of auxotrophic strains of lactic acid bacteria to pyridoxine led to the discovery, synthesis, and demonstration of the natural occurrence and vitamin B, activity of pyridoxal and pyridoxamine (TABLE1). Although pyridoxine and vitamin B, are still frequently used as synonyms, especially by medical researchers, this practice is erroneous and sometimes misleading; vitamin B, is a complex comprised of three chemically different compounds and their combined forms. Coincident with these developments, the next chapter in the vitamin B, story was being written by enzymologists and is summarized in TABLE2. Detailed study of inducible bacterial amino acid decarboxylases by Gale and his colleagues resulted in the discovery that these enzymes required a new coenzyme. Independently, Gunsalus et al., using the tyrosine decarboxylase in cells of Streptococcus faecalis as a test system, showed that this coenzyme was a phosphorylated pyridoxal, which was eventually shown by unequivocal methods of synthesis to be pyridoxal 5’-phosphate (PLP, TABLE2). The same coenzyme, PLP, was later shown to be required for the action of tryptophanase, aspartate aminotransferase, and a variety of other enzymatic 1
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TABLE 1. Discovery and Nature of the Vitamin
1934 1938 1939 1942-44 1944 1945-46
Vitamin B6 defined by “specific” rat assay: P. Gyiirgy (B6), S. Lepkovsky (Factor 1). Isolation of pyridoxine: Lepkovsky (Feb., rice bran); Keresztesy & Stevens (Feb., rice bran); Gyiirgy (April, Peter’s eluate [yeast]); Kuhn & Wendt (May, yeast); Ichiba & Michi (rice bran). Proof of structure and synthesis of pyridoxine: Stiller, Keresztesy & Stevens; Harris & Folkers, Kuhn & Wendt. “Pseudopyridoxine” (pyridoxal + pyridoxamine) discovered as vitamins for lactic acid bacteria: Snell; Snell, Guirard & Williams, Carpenter & Strong. Synthesis and activity of pyridoxal and pyridoxarnine: Harris, Heyl & Folkers; Snell. Natural occurrence and comparative activities of pyridoxal, pyridoxamine and pyridoxine for bacteria, fungi, and animals: Snell; Snell & Rannefeld; Sarma, Snell& Elvejem; Luckey et al.
‘For references, see Snell.’
transformations of amino acids. These early findings established PLP and (for the transaminases) P M P as the functional coenzyme forms of vitamin B,.
VARIATIONS IN COFACTOR REQUIREMENTS OF HISTIDINE DECARBOXYLASES The effects of PLP on partially purified preparations of many of the known decarboxylases are shown in TABLE3. Even in these crude preparations the requirement for this coenzyme is obvious. However, in two cases, the glutamate decarboxylase from Escherichia coli and the histidine decarboxylase (HisDCase) from Clostridium welchii, no stimulation by PLP was evident, and Gale suggested that PLP was not a coenzyme for these enzymes. This suggestion was disproved for glutamate decarboxylase by Shukuya and Schwert; who first isolated the homogeneous enzyme and showed that it contained TABLE 2. Discovery and Nature of Coenzyme Forms of Vitamin
1940-45 1944 1944 1945-47 1947 1951-52 1954-57
Inducible bacterial amino acid decarboxylases (Arg, Lys, Om, Tyr) require an unidentified coenzyme: Gale, Epps & coworkers. Formation of tyrosine decarboxylases in S . faecalis requires “pseudopyridoxine”: Bellamy & Gunsalus. Coenzyme for tyrosine decarboxylase is a phosphorylated pyridoxal, probably pyridoxal S’-phosphate (PLP): Gunsalus, Bellamy & Umbreit. PLP established as coenzyme for transamination, trytophanase, etc.: Snell; Schlenk & Snell; Lichstein, Umbreit & Gunsalus; Wood & Gunsalus. Pyridoxamine phosphate (PMP) also occurs naturally: Rabinowitz & Snell. Unequivocal synthesis of PL-5’-P and PM-5’-P, the coenzyme forms of vitamin B,: Heyl et al.; Baddiley & Matthias. PMP also acts as coenzyme for transaminases: Meister, Sober & Peterson; Jenkins & Sizer.
‘For references see Snell.1-3
SNELL: B, AND DECARBOXYLATION OF HISTIDINE
stoichiometric amounts of firmly bound PLP. Since then, many amino acid decarboxylases have been isolated as homogeneous PLP-dependent enzymes: All contain 2 to 12 identical subunits, with one PLP per subunit. For HisDCase, reality is more complex. Crude preparations of the mammalian enzyme were stimulated by PLP, but partially purified preparations from C. welchii (TABLE3 ) , Lactobacillus 30a. and Micrococcus sp. nov. were not. Work in my laboratory has now clarified these seeming discrepancies. We have purified HisDCase to homogeneity from both L. 30a’ and from C. perfringens (welchii).’ Neither enzyme contains PLP. The spectrum of the L. 30a enzyme is that of a simple protein (FIG. 1A) with no trace of absorbance characteristic of PLP in the 41 5-430-nm region. Nevertheless, the enzyme is inhibited by carbonyl reagents, and Dixon Riley and I showed” that the reactive carbonyl group was present in a covalently bound pyruvate residue. By contrast, the spectrum of homogeneous HisDCase purified from Morgunelfa morganii is typical of a PLP enzyme with an absorbance at 420 nm about 10% that at 280 nm (FIG. 1B).
TABLE 3. PLP Requirements for Crude Preparations of Amino Acid Decarboxylases
Substrate L-Lysine L-Tyrosine L- Arginine L-Ornithine L-Glutamate L-Histidine
meso-2,6-Diamino-pimelate Nonspecific (valine, leucine, etc.) Aromatic amino acid decarboxylase
Source E. coli S. faecalis E. coli E . coli E. coli Mammals C. welchii Mamma 1s E. coli P. vulgaris Mammals
rrl CO,Liberated/S min + PLP Alone
195 174 118 110
20 10 20
25 no resolution -
+++ +++ +++ +++
aAdapted from Gale4and Guirard and Snell.’
Two types of HisDCase thus exist: One type neither contains nor requires PLP as coenzyme, but instead contains pyruvate as an essential, covalently bound prosthetic group; the second type is a typical PLP-dependent decarboxylase. So far, the HisDCase from each of four gram-positive organisms has been a pyruvoyl enzyme, while that from each of three gram-negative organisms has proved to be PLP-dependent (TABLE 4). Individual HisDCases of a single type resemble one another closely, but differ greatly from enzymes of the other class not only in their prosthetic group but also in subunit structure, pH optimum, biogenesis, sensitivity to inhibitors, and other properties (TABLE5). They are, however, similar in catalytic efficiency. These proteins thus present a rare example of independent evolution of completely different but functionally identical enzymes, and provide an unusual opportunity to compare at the molecular level the mechanisms of two different solutions to a single problem, the decarboxy-
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FIGURE 1. Spectrum of homogeneous HisDCase from: (A) Lactobacillus 300.' and (B) Morganella morganii? In A, curves 1 and 2 represent, respectively,spectra at pH 8.5 and 4.8; in B, solid and dotted lines represent, respectively, spectra before and after reduction with NaCNBH,.
Iation of histidine. Clarification of these mechanisms is the ultimate goal of our current work on HisDCase. I will next describe briefly our progress toward this goal by comparing a wellstudied representative from each of these two classes of HisDCases: the PLP enzyme from M . rnorgunii, and the pyruvoyl enzyme from L. 30a.
THE PLP-DEPENDENT HISDCASEOF M . MORGANZZ The general properties of this HisDCase have been presented earlier (TABLE5). The problem of mechanism can be considered in two overlapping parts: the role of (a) the coenzyme, and (b) the apoenzyme.
The Role of PLP The role of P L P has been considerably clarified through study of nonenzymatic model reactions and various PLP-dependent enzyme reactions. Present concepts of this TABLE 4. Correlation of Type with Source of Histidine Decarboxylase
Pyruvoyl-Dependent".' Lactobacillus 30a Lactobacillus buchneri Clostridium perfringens Micrococcus sp. nov.
PLP-Dependent Morganella morganii' Klebsiella plantico1a'2 Enterobacier aerogenes"
Fetal rat l i ~ e r ' ~ . ' ~ Mouse kidney''
SNELL: B6 AND DECARBOXYLATION OF HISTIDINE
Comparison of PLP- (from M . morgunii) and Pyruvoyl-Dependent (from L. 30u) Histidine Decarboxylases
HisDCase from M . mor~aniiqJ2 M , of native enzyme Subunit structure M , of a subunit M , of ,@ subunit PLP per subunit Pyruvate per subunit pH optimum K , at pH optimum Vmax,pmol min-' m g I Turnover no./active site min
- 171,000 a4
42.742' 1 0 6.5 1.1 150 -6,400
204,600' ( 4 1 6
25,260' 8,840' 0 1 (aonly) 4.8 0.4 60-80 -2,700
'Calculated from the amino acid sequence."'^"
role are presented in FIGURE 2. PLP is present in HisDCase, as in other enzymes, as an internal aldimine formed with the amino group of a specific lysine residue. This internal aldimine group reacts with the amino acid substrate by transaldimination to form an external aldimine (the ES complex) which, by virtue of the strongly electrophilic character of its pyridoxylidene moiety, weakens the bond to the carboxyl group, resulting in loss of CO,. A proton adds to the resulting carbanion, and the product is released. Work with both enzymatic and nonenzymatic models has confirmed (a) that direct labilization of the C L C O O H bond occurs (the a-H is not labilized, and
FIGURE 2. Present concepts of the role of PLP in decarboxylation of amino acids. (From Boecker & Snell.I8 Used with permission.)
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a-methyl amino acids are decarboxylated); (b) that free CO,, not HCO;, is evolved; and (c) in enzymatic reactions the incoming proton assumes the stereochemical position vacated by the leaving COOH group. All double bonds of the transition state are thought to lie in a plane that facilitates the indicated electron displacements; for efficient labilization in such a system the bond to the carboxyl group must lie perpendicular to this lane.'^.'^
The Role of the Apoenzyme In the scheme of FIGURE 2 the protein obviously provides, in addition to the specific lysine residue that interacts with PLP, other specific binding sites for PLP and For the substrate that properly orients the two for reaction, as well as catalytic residues that greatly enhance the reaction rate. To obtain information about the nature of these groups, we have cloned and sequenced the gene for this HisDCase” and expressed it in E. coli; the expressed protein is identical with the wild-type enzyme. The gene-encoded peptide chain (FIG.3) contains 377 amino acid residues, corresponding to one subunit of the mature enzyme. Lys 232 provides the a-amino group that forms the internal aldimine with PLP, while Ser 322 is the residue destroyed during the mechanism-based inactivation of HisDCase by a-fluoromethylhistidine.The sequence,-Ser-X-His-Lysthat includes Lys 232 appears in this and three other decarboxylases, a possible indication of its catalytic importance.”*’* These considerations prompted my colleague, Gloria Vaaler, to prepare sitespecific mutant HisDCases modified at each of the positions indicated by the arrows at the top of TABLE6. The activity of these mutant proteins (TABLE6) leads to several
ERFLRFSINNCGDWGEY CNYLLNSFDFEKEVMEYFADL 77
FKIPFEQSWGYVTNGGTEGNMFGCY LGREIFPDGTLYYS 116
KDTHY SVAKIVKLLRIKSQVVESQPNGEIDYDDLMKKIA 155 DDKEAHPIIFANIGTTVRGAIDDIAEIQKRLKAAGIKRED
195 * YYLHADAALSGMILPFVDDAQPFTFADGIDSIGVSGHKM 234 IGSPIPCGIVVAKKENVDRISVEIDYISAHDKTITGSRNC
275 TPLMLWEAIRSHSTEE WKRRITRSLDMAQY AVDRMQKA 313
GINAWRNKNSITVVFPCPSERVWREHCLATSGDVAHLIT 352 TAHHLDTVQIDKLIDDVIADFNLHAA FIGURE 3. Amino acid sequence of one subunit of the PLP-dependent HisDCase of M . morganii.” Residue 232 (*) forms an internal azomethine link with P L P both the bound PLP residue and residue 322 (+) react with a-fluoromethyl histidine during mechanism-based inactivation of the enzyme with this inhibitor.
SNELL B, AND DECARBOXYLATION OF HISTIDINE TABLE 6.
Specific Activity of Mutant Histidine Decarboxylasesa
229 Wild type S229A H231R H23 1F H231Q H231N
127 10 0 0 15
K232A M233I C240A S322A C329A C329S
0 38 95 41 2.4 34
‘KisDCase in the soluble fraction frum cells induced at 25°C was determined by quantitative densitometry of SDS-PAGE gels. Assays were at 3 7 T . Specificactivity = pmol CO, min- Img-’ (from Vaaler & SneP).
conclusions. (a) Modification of any of these residues reduces activity, but since one or more of the mutant enzymes with replacements at Ser 229, His 231, Met 233, Cys 240, Ser 322, or Cys 329 show substantial activity, these residues are not catalytically essential. Most probably, their replacement decreases activity through effects on enzyme conformation that result in suboptimal binding of PLP or histidine, or a slightly distorted orientation of groups necessary for catalysis, but the true explanation will require further study of the purified proteins. (b) His 231 seems to participate in formation of a critical hydrogen bond via the 7-N of its imidazole ring. This conclusion follows from (i) the inactivity of the His231Phe protein, which contains a residue of similar size; (ii) the substantial activity of the His231Gln protein, in which the amide N of the Gln residue (which can also hydrogen bond) occupies a position spatially equivalent to the 7-N of the imidazole group of His 231; and (iii) the very low activity of the His231Asn protein, with its amide group on a shorter side chain. (c) Replacement of Lys 232 with Ala gives an inactive protein. We have purified this protein to homogeneity: Like the wild-type enzyme, it binds one PLP per subunit, but does not absorb at 420 nm, where an internal aldimine would absorb (cf. FIG.1B). This PLP protein binds histidine as well as the wild-type enzyme, but decarboxylates it a t a rate less than 0.0002 times the rate at which it is catalyzed by the wild-type enzyme (Vaaler & Sne1lzo). We believe these data indicate a critical catalytic role for the Lys 232 residue of HisDCase, probably as the proton donor required for the decarboxylation reaction.
THE PYRUVOYL-DEEPENDENT HISDCASE OF L. 30A Since discovery of the pyruvoyl-dependent HisDCase of L. 3#a, several similar decarboxylases (see TABLE 7) have been found that act on other substrates. All contain pairs of two dissimilar subunits; in all cases the essential pyruvoyl group blocks the amino terminus of one subunit, as shown for HisDCase in FIGURE4. The presence of covalently bound pyruvate raises two questions: (a) what is its origin, and (b) how does it function? I shall restrict discussion of these topics to the HisDCase of L. 30a with the implicit assumption (yet to be fully validated) that similar explanations will hold true for other enzymes of this group.
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TABLE 7. Pyruvoyl-Dependent Amino Acid Decarboxylases
Subunits, M, x lo-'
Histidine decarboxylases'' Lactobacillus 30a L. buchneri C.perfringens Micrococcus sp. nov. S-Adenosylmethionine decarboxylases E . coli2'.22
Mammalian2' Yeast24 Phosphatidylserine decarboxylase E. ~ o l i ~ ~ Aspartate a-decarboxylase'' E . coli
25 25 25 29
9 9 9 8
( 4 6
(01fl)6 ( 4 5
Origin of the Pyruvoyl Group: proHisDCase Isotopic labeling studies showed that the pyruvoyl group of HisDCase arises without dilution from serine. The immediate precursor, proHisDCase, accumulates in mutant three of L. 30a and is readily isolated. It contains no pyruvate and only one type of subunit (the 7~ subunit). At pH 7.5 it self-activates by an overall reaction (FIG. 5) that generates ammonia, a pyruvate residue (from Ser 82 of the precursor T chain), and the a and 6 chains of the active enzyme without loss of any amino acids." When activation was conducted in H,"O, no "0was incorporated into the carboxyl-terminal serine (Ser 81) of the 0 subunit, showing that activation did not involve hydrolytic cleavage of the peptide bond. However, during activation of appropriately labeled proenzyme in H,l60, "0was transferred quantitatively from the hydroxyl group of Ser 82 to the carboxyl group of Ser 8 1. Two reaction pathways that might accomplish this are shown in FIGURE 6. More recent results have shown that activation cannot proceed by pathway 11, because we have prepared its unsaturated key intermediate by
Tyr ( a - c h a i n )
Ser ( % - c h a i n )
Se r in.W.
N o / n a t i v e enzyme
(m.w. 2 0 8 ,000)
6 p H 7.0,