2,3-DIHYDROXYBENZOIC ACID DECARBOXYLASE FROM ASPERGILLUS-NIGER - A NOVEL DECARBOXYLASE

2,3-Dihydroxybenzoic acid decarboxylase, the last enzyme in the fungal metabolism of indole to catechol, catalyzes the non-oxidative decarbo xylation of 2,3-dihydroxybenzoic acid to catechol. Unlike most other d ecarboxylases, this enzyme does not require a cofactor, underlining th e importance of active-site residues in the reaction mechanism. Earlie r studies from this laboratory [Kamath, A. V., Appaji Rao, N. & Vaidya nathan, C. S. (1989) Biochem. Biophys. Res, Commun. 165, 20-26], have shown that the sulfhydryl agent N-ethylmaleimide (MalNEt) inactivated the enzyme by modifying a single class of cysteine residues and that t his inactivation was prevented in the presence of salicylate, a substr ate analogue. in the present study, this essential cysteine residue ha s been identified by specific labelling with [C-14]-MalNEt using the d ifferential labelling technique. The stoichiometry of incorporation of [C-14]MalNNEt was approximately one/subunit of the homotetrameric pro tein. The peptide bearing this reactive cysteine residue was isolated by tryptic digestion of the differentially labelled enzyme and subsequ ent reverse-phase chromatography of the peptide mixture. The sequence of the major radioactive peptide that was identified to be the active- site peptide, was LLGLAETCK. A search for sequences similar to this ac tive-site peptide indicated that this sequence was probably unique to the decarboxylase under study. A partial primary structure map constru cted from the sequences of peptides derived from enzymic cleavage of t he protein using endoproteinase Glu-C and trypsin did not share any si gnificant sequence similarity with sequences reported in the database, again suggesting the uniqueness of the enzyme. This is the first repo rt on the active-site peptide and the partial primary structure of a n on-oxidative decarboxylase catalyzing the removal of a carboxyl group from an aromatic nucleus.