Cloning, sequencing, expression, and site-directed mutagenesis of the gene from Clostridium perfringens encoding pyruvoyl-dependent histidine decarboxylase.
The DNA encoding pyruvoyl-dependent histidine decarboxylase (HisDCase) of Clostridium perfringens was cloned, sequenced, and overexpressed in Escherichia coli. The gene encodes a single polypeptide of 320 amino acids, Mr 35,526, demonstrating that clostridial HisDCase, which has an (alpha beta)6 structure, is synthesized as a precursor (proHisDCase, pi 6). No pi subunits of proHisDCase were observed in crude or purified preparations of the cloned HisDCase; they appear to undergo rapid cleavage in vivo to the alpha (Mr 24,887) and beta (Mr 10,526) subunits characteristic of this HisDCase. This cleavage occurs between Ser-96 and Ser-97; Ser-97 gives rise to the catalytically essential pyruvoyl group blocking the N-termini of the alpha subunits of the active enzyme. When Ser-97 was converted to an alanyl residue by site-specific mutagenesis, the expressed, inactive protein (pi' 6) contained a single peptide species (pi', Mr 35,510) that was not cleaved either in vivo or in vitro. These results support previous conclusions that activation of the wild-type clostridial proenzyme occurs via nonhydrolytic serinolysis. Although clostridial HisDCase has only a 47% sequence similarity to HisDCase from Lactobacillus 30a, all of the residues known to be important for substrate binding and catalytic action of the Lactobacillus HisDCase are conserved in the C. perfringens enzyme. While the encoded N-terminal Met of clostridial HisDCase is removed by E. coli, the cloned enzyme retains a 10-residue presequence (NKNLEANRNR) not present in the mature enzyme isolated from C. perfringens.
van Poelje
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Department of Chemistry, University of Texas, Austin 78712.
Snell
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D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000235:genetics*; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000235:genetics; D004792:Enzyme Precursors / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D006640:Histidine Decarboxylase / Q000235:genetics* / Q000302:isolation & purification; D007778:Lactobacillus / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics* / Q000302:isolation & purification; D012689:Sequence Homology, Nucleic Acid; D012694:Serine / Q000235:genetics
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D012694:Serine; D002262:Carboxy-Lyases; D006640:Histidine Decarboxylase; D000409:Alanine