Sequence of ornithine decarboxylase from Lactobacillus sp. strain 30a.

A gene encoding biodegradative ornithine decarboxylase from Lactobacillus sp. strain 30a was isolated from a genomic DNA library and sequenced. Primer extension analysis revealed two transcription initiation sites. The deduced amino acid sequence is compared with the amino acid sequences of five previously reported bacterial decarboxylases, and conserved pyridoxal phosphate motif residues are identified.

Pyridoxal phosphate-dependent histidine decarboxylases. Cloning, sequencing, and expression of genes from Klebsiella planticola and Enterobacter aerogenes and properties of the overexpressed enzymes.

The hdc genes encoding the inducible pyridoxal-P-dependent histidine decarboxylase (HisDCase) of Klebsiella planticola and Enterobacter aerogenes were isolated, sequenced, and expressed in Escherichia coli under control of the lac promoter, and the overproduced enzymes were purified to homogeneity from the recombinant host. Formation of inclusion bodies during synthesis of the E. aerogenes enzyme was avoided by cooling the culture and inducing at 25 degrees C. The cloned enzymes were produced in amounts three to four times those present in the fully induced native hosts and were identical in properties to those isolated earlier (Guirard, B. M., and Snell, E. E. (1987) J. Bacteriol. 169, 3963-3968). The two enzymes showed 85% sequence identity and also showed 80% sequence identity with the previously sequenced (Vaaler, G. L., Brasch, M. A., and Snell, E. E. (1986) J. Biol. Chem. 261, 11010-11014) HisDCase of Morganella morganii. Nevertheless, antibodies to the M. morganii HisDCase do not cross-react with these enzymes suggesting that the regions of amino acid variations are located on the outer surface of the proteins. All three HisDCases are the same length (377 amino acid residues); encoded N-terminal methionine was completely removed in each case. These closely related pyridoxal-P enzymes show no sequence homology with the pyruvoyl-dependent HisDCases of Gram-positive bacteria.

Pyridoxal 5'-phosphate dependent histidine decarboxylase: overproduction, purification, biosynthesis of soluble site-directed mutant proteins, and replacement of conserved residues.

The hdc gene coding for the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii has been expressed in Escherichia coli under control of the lac promoter. The enzyme accumulates to 7-8% of total cell protein and is purified to homogeneity by passage through three columns. Fourteen site-directed mutant enzymes were constructed to explore the roles of residues of interest, especially those in the sequence Ser229-X230-His231-N epsilon-(phosphopyridoxylidene)Lys232, since identical sequences also appear in several other decarboxylases. Most of the overproduced mutant proteins were aggregated into inclusion bodies, but when the late log phase cultures were cooled from 37 to 25 degrees C before induction, the mutant proteins were obtained as soluble products. Ala or Cys in place of Ser-229 yielded mutant enzymes about 7% as active as wild-type, indicating that this serine residue is not essential for catalysis but contributes to activity through conformational or other effects. Of the replacements made for His-231 (Asn, Gln, Phe, and Arg), only Gln and Asn gave partially active enzymes (about 12% and 0.2% of wild-type, respectively). The side-chain amide of Gln may act by mimicking the positionally equivalent tau-nitrogen on the imidazole ring of histidine to provide an interaction (e.g., a hydrogen bond) required for efficient catalysis. The Lys-232 residue that interacts with pyridoxal 5'-phosphate appears central to catalytic efficiency since replacing it with Ala yields a mutant protein that is virtually inactive but retains the ability to bind both pyridoxal 5'-phosphate and histidine efficiently.(ABSTRACT TRUNCATED AT 250 WORDS)

Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Nucleotide sequence of the hdc gene and the corresponding amino acid sequence.

The nucleotide sequence of a 1.3-kilobase NaeI fragment from Morganella morganii AM-15 that contains the gene for histidine decarboxylase has been determined. The gene was initially identified among total chromosomal digests using a mixed sequence oligonucleotide probe corresponding to amino acids 11-16 of histidine decarboxylase and then cloned on a 5.5-kilobase PstI fragment. The structural gene contains 1131 nucleotides and encodes 377 amino acids with the sequence: (sequence: in text). The independently determined NH2-terminal sequence of this enzyme (Tanase, S., Guirard, B. M., and Snell, E. E. (1985) J. Biol. Chem. 260, 6738-6746) and the amino acid sequences of two tryptic peptides reported in the accompanying paper (Hayashi, H., Tanase, S., and Snell, E. E. (1986) J. Biol. Chem. 261, 11003-11009) are localized in the sequence presented here; the lysine that binds pyridoxal phosphate is situated at residue 232, whereas the serine that binds the adduct formed between pyridoxal phosphate and the inhibitor alpha-fluoromethylhistidine is positioned at residue 322.

Histidine decarboxylase of Lactobacillus 30a. Sequences of the cyanogen bromide peptides from the alpha chain.

The alpha chain of histidine decarboxylase contains eight internal methionine residues. After reductive amination to convert the NH2-terminal pyruvoyl residue to an alanyl residue and cyanogen bromide treatment, 13 pure peptides were isolated. Four of these are incomplete cleavage products. The sequence of each of the remaining nine peptides was established by automated and manual degradation of the intact peptides and of smaller peptides obtained from tryptic, chymotryptic, and staphylococcal protease digests of the cyanogen bromide peptides. These results, together with the data on overlapping peptides reported in the accompanying paper (Huynh, Q. K., Recsei, P. A., Vaaler, G. L., and Snell, E. E. (1984) J. Biol. Chem. 259, 2833-2839), establish the complete amino acid sequence of the alpha chain.

Histidine decarboxylase of Lactobacillus 30a. Sequences of the overlapping peptides, the complete alpha chain, and prohistidine decarboxylase.

The complete amino acid sequence of the alpha chain of histidine decarboxylase of Lactobacillus 30a has been established by isolation and analysis of the eight methionine-containing tryptic peptides of this chain. These peptides provide the overlaps required to order all nine peptides derived by complete cyanogen bromide cleavage of the alpha chain (Huynh, Q.K., Vaaler, G.L., Recsei, P.A., and Snell, E.E. (1984) J. Biol. Chem. 259, 2826-2832). Ordering of six of the latter peptides was confirmed by isolation and analysis of four peptides derived by incomplete cyanogen bromide cleavage. The alpha chain is composed of 226 residues and has a molecular weight of 24,892 calculated from the sequence. These results and the previously determined sequence of the beta chain (Vaaler, G.L., Recsei, P.A., Fox, J.L., and Snell, E.E. (1982) J. Biol. Chem. 257, 12770-12774) establish the complete amino acid sequence of the enzyme and of the pi chain of prohistidine decarboxylase. The latter is composed of 307 amino acids and has a calculated molecular weight of 33,731. Four segments of the pi chain sequence are repeated. The bond between Ser-81 and Ser-82 that is cleaved during proenzyme activation is in an uncharged portion of the sequence that is rich in serine and threonine residues and is predicted to be part of a beta sheet structure.

Histidine decarboxylase of Lactobacillus 30a. Comparative sequences of the beta chain from wild type and mutant enzymes.

Manual and automated sequencing of peptides isolated from tryptic, chymotryptic, and Staphylococcus aureus V8 protease digests of the beta chain of histidine decarboxylase from Lactobacillus 30a have established the following sequence for the wild type protein: NH2-Ser-Gly-Leu-Asp-Ala-Lys-Leu-Asn-Lys-Leu-Gly-Val-Asp-Arg-Ile-Ala-Ile-Ser-Pro -Tyr-Lys-Gln-Trp-Thr-Arg-Gly-Tyr-Met-Glu-Pro-Gly-Asn-Ile-Gly-Asn-Gly-Tyr-Val-Thr-Gly-Leu-Lys-Val-Asp-Ala-Gly-Val-Arg-Asp-Lys-Ser-Asp-Asp-Asp-Val-Leu-Asp-Gly-I le-Val-Ser-Tyr-Asp-Arg-Ala-Glu-Thr-Lys-Asn-Ala-Tyr-Ile-Gly-Gln-Ile-Asn-Met-Thr- Thr-Ala-Ser-COOH The beta chain from mutant 3 of this organism shows two amino acid replacements: Ser-51 is replaced by Ala and Gly-58 by Asp. These amino acid replacements result in a significant increase in the predicted alpha-helical content and a significant decrease in the isoelectric point of the mutant beta chain and are consistent with changes in physical and catalytic properties of the mutant histidine decarboxylase. In addition, about 15% of the mutant chains contain Met-Ser at the NH2 terminus rather than Ser. Asn-35 is partially deamidated in both proteins. Structural comparisons show that the histidine decarboxylase from Lactobacillus 30a and a similar pyruvoyl enzyme from Micrococcus sp. n. have evolved from a common ancestral protein.