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 decarboxylation of 2,3-dihydroxybenzoic acid to catechol. Unlike most other decarboxylases, this enzyme does not require a cofactor, underlining the importance of active-site residues in the reaction mechanism. Earlier studies from this laboratory [Kamath, A. V., Appaji Rao, N. & Vaidyanathan, 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 this inactivation was prevented in the presence of salicylate, a substrate analogue. In the present study, this essential cysteine residue has been identified by specific labelling with [14C]-MalNEt using the differential labelling technique. The stoichiometry of incorporation of [14C]MalNEt was approximately one/subunit of the homotetrameric protein. The peptide bearing this reactive cysteine residue was isolated by tryptic digestion of the differentially labelled enzyme and subsequent 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 active-site peptide indicated that this sequence was probably unique to the decarboxylase under study. A partial primary structure map constructed from the sequences of peptides derived from enzymic cleavage of the protein using endoproteinase Glu-C and trypsin did not share any significant sequence similarity with sequences reported in the database, again suggesting the uniqueness of the enzyme. This is the first report on the active-site peptide and the partial primary structure of a non-oxidative decarboxylase catalyzing the removal of a carboxyl group from an aromatic nucleus.
Santha
R
R
Department of Biochemistry, Indian Institute of Science, Bangalore.
Savithri
H S
HS
Rao
N A
NA
Vaidyanathan
C S
CS
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D001665:Binding Sites; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000502:physiology; D002396:Catechols / Q000378:metabolism*; D062385:Hydroxybenzoates / Q000378:metabolism*; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship
D002396:Catechols; D062385:Hydroxybenzoates; C009135:2,3-dihydroxybenzoic acid; D002262:Carboxy-Lyases; C034221:catechol