Pyruvoyl-dependent Histidine Decarboxylases PREPARATION AND AMINO ACID SEQUENCES OF THE j3 CHAINS OF HISTIDINE DECARBOXYLASE FROM CLOSTRIDIUM PERFRINGENS AND LACTOBACILLUS BUCHNERI*

Histidine decarboxylase (HisDCase) from Lactoba- cillus buchneri was purified to homogeneity. Its subunit structure, (afl)s, and enzymatic properties resem- ble closely those of the immunologically cross-reactive HisDCase of Lactobacillus 30a (Recsei, P. A., and Snell, E. E. (1984) Annu. Rev. Biochem. 53,357-387). The complete amino acid sequences of the fl chains of the HisDCase from L. buchneri (81 residues) and Clostridium perfringens (86 residues) were then deter- mined to be a and b, respectively.


V L D G I G S Y D R A E T K N A Y I G Q I N M T T A S (b) T L S E G I H K N I K V R A P K I D K T A I S P Y D R Y C D G Y G M P G A Y G D G Y V S V L K V S V G T V K K T D D I L L D G I V S Y D R A E I N D A Y V G Q I N M L T A S
Although these sequences differ substantially near the NH2-terminal ends, there is striking homology near the COOH termini and also near the NHa terminus of the two a chains (pyruvoyl-Phe-X-Gly-Val-, where X is Ser or Cys). If the four known pyruvoyl-dependent HisDCases arise from inactive proenzymes by the mechanism previously demonstrated for the HisDCase of Lactobacillus 30a (Recsei, P. A., Huynh, Q. K. and Snell, E. E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 973-977), then each of these proenzymes has the sequence -Thr-Ala-Ser-Ser-Phe-at the activation site (where -Serbecomes the COOH terminus of the fl chain and -Serbecomes the pyruvoyl group blocking the NH2 terminus of the a chain), and the sequences around this activation site are highly conserved in all four enzymes. These facts support the assumptions that the four enzymes have evolved from a common ancestral protein, are formed from inactive pyruvate-free proenzymes by similar mechanisms, and have similar catalytic mechanisms.
Four pyruvoyl-dependent HisDCases' are now known (1). All of them have an (a@, subunit structure, with the essential pyruvoyl group blocking the NH2 terminus of the larger, a, subunit. The HisDCase of Lactobacillus 30a has been most * This work was supported in part by Grants AI 13940 and AM 19898 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The abbreviations used are: HisDCase, histidine decarboxylase; HPLC, high performance liquid chromatography; TPCK, L-l-tosylamido-2-phenylethyl chloromethyl ketone; PN, the pyruvoyl residue; PTH-amino acid, phenylthiohydantoin of a given amino acid. extensively studied. It has an (aj3)6 structure that arises by the intramolecular nonhydrolytic cleavage of an inactive proenzyme, ?TI39 coupled with formation of the essential pyruvoyl group from a serine residue at the cleavage site (1, 2).
The complete sequences of the T , a, and 0 chains are known (3,4). To assess the generality of this so far unique mechanism of proenzyme activation, as well as the sequence requirements for enzymatic activity, the sequences surrounding the active site of other pyruvoyl-dependent HisDCases would be of interest. We describe here the purification to homogeneity of HisDCase from Lactobacillus buchneri and some of its properties, the complete sequence of the j3 chains of HisDCases from L. buchneri and from Clostridium perfringens, and an extended NH2-terminal sequence of the a chains from these same two enzymes. Together with published data (3-5) these results show that although major differences in sequence occur elsewhere, all four of the known HisDCases show strikingly similar sequences around the active pyruvoyl residue and hence around the activation site of their putative proenzymes.
EXPERIMENTAL PROCEDURES AND RESULTS~ DISCUSSION HisDCase from L. buchnerii is cross-reactive with antibodies to HisDCase from Lactobacillus 30a, but the two enzymes are not identical (6). Immunoprecipitates of the L. buchneri enzyme were used previously to establish that this enzyme like that from Lactobacillus 30a has an (a@)x structure, contains a pyruvoyl group on the a subunit, and is formed from a proenzyme, A= (1,6). We have now purified this enzyme to homogeneity by a more conventional 4-step procedure (Table  11, Miniprint). It has an (aB)e structure and closely resembles the Lactobacillus 30a enzyme in its catalytic parameters (Table 111,Miniprint). This preparation of the L. buchneri enzyme and a homogeneous sample of the HisDCase from C. perfringens prepared as described previously (6) were then used to prepare the separate a and 0 subunits from each enzyme for comparative sequence studies.
The complete amino acid sequences of the j3 chains of HisDCases from C. perfringens and L. buchneri are shown in .

T 3 o -
"--I I , , =-= " T I -  (+) indicate identification by HPLC of phenylthiohydantoins following automated sequencing. Half-arrows (-) indicate identification by manual sequencing. Reverse arrows (c) indicate identification by carboxypeptidase digestion. Data printed above the sequences are from steps performed on the intact proteins and those below the sequences are from steps performed on the digestion products.

perfringens (A) and L. buchneri (B). FuU arrows
Peptides are designated T, tryptic; C, chymotryptic; and CB, cyanogen bromide. those previously estimated from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (6). The amino acid compositions determined from the sequences also are in good agreement with those obtained by amino acid analyses (Table IV, Miniprint).
One Met-Thr sequence occurs in the L. buchneri /3 chain at positions 77-78. Such a sequence has previously been reported to be resistant to CNBr (7) and was cleaved in the L. buchneri / 3 chain only to the extent of about 15% (Table XIV, Mini-print). There were, in contrast, some chymotryptic and subtilisin-like cleavages during digestion of the / 3 chains of both enzymes with trypsin (peptide T3b of the L. buchneri His-DCase and peptide T1 of the C. perfringens (HisDCase, Fig.   1). Such autolytic development of chymotryptic and subtilisin activities during tryptic digestion has been reported previously (8-10). The other unexpected result was our failure to find the NHz-terminal peptide (Thr-Leu-Ser) of the C. perfringens /3 chain during purification of the tryptic peptides by HPLC.  This peptide sequence, however, was determined by analysis of the NHn-terminal sequence on the intact j3 chain (Table  IX) and confirmed by the sequences of chymotryptic peptide C1 and cyanogen bromide peptide CB1 (Fig. LA). Only singleresidue overlaps of isolated peptides are shown in Fig. 1 for the j3 chain of the C. perfringens enzyme at residues 27-28 and 47-48 and for that of the L. buchneri enzyme at residues 20-21 and 42-43. These positional assignments are further supported by the results of automated sequencing from the NH2 terminus of the intact j3 chains (Tables IX and XI, M i n i p r~t~ and by the amino acid compositio~ of cyanogen bromide peptides, CB1 and CB2 (Table X and XIV, Miniprint). A comparison of the sequences of the j3 chains from four sources is shown in Fig. 2, which also compares the NH2terminal sequences of the CY chains from these same four sources. The L. buchneri j3 chains shows high homology (86%) with the j3 chain of the Lactobacillus 30a enzyme, but there is much less homology among the j3 chains of HisDCases from C. perfringens, LactobaciUus 30a and Micrococcus sp. n. (Table  I). These results are consistent with the findings that rabbit antibodies prepared against HisDCase from Lactobacillus 30a cross-react with HisDCase from L. buchneri but not with that from C. ~r f r i~e~ (6). Only 23% of the @ chain residues are identical in all four enzymes, and most of these residues are concentrated near the COOH-terminal portion of the j3 chain (Fig. 2). Similarly, residues near the essential pyruvoyl residue at the NH, terminus of the a chain are more highly conserved than those further removed (Fig. 2). In proHisDCase from Lactobacillus 30a the carboxyl-terminal serine (Ser) of the j3 chain and the serine precursor (Ser) of the a chain pyruvoyl residue are linked in the proenzyme sequence, -Thr-Ala-Ser-Ser-Phe-, that overlaps the COOH terminus of the j3 chain (-Thr-Ala-Ser) and the NH2 terminus (Prv-Phe-) of the a chain (3, 11). Putative proenzymes for each of the four HisDCases would have this Bame sequence, with highly conserved sequences on either side (Fig. 2). Although such proenzymes have been detected so far only in LactobaciUus 30a (12) and L. buchneri (6), these considerations, together with the common properties described in Table 111, indicate that the four enzymes have evolved from a common ancestral protein, that they are formed from inactive pyruvate-free proenzymes by a mechanism similar to that described for the lac to^^ 30a enzyme (21, and that active sites in the mature enzyme and activation sites in the postulated proenzymes are conformationally similar with probably similar catalytic residues. Acknowledgment-We are indebted to Michiko S. Huynh for assistance in growing, harvesting, and acetone drying the cells of C. perfringens.