Biochemical characterisation of an aldoxime-forming flavoprotein involved in 2-phenylethylglucosinolate biosynthesis in Brassica species

L-Homophenylalanine (L-HPhe) is the precursor of 2-phenylethylglucosinolate, a secondary metabolite present in some Brassica and related species. A key step in its biosynthesis is the oxidative decarboxylation of L-HPhe to its aldoxime. The enzyme catalysing this reaction has been shown to be a NADPH- and O-2-dependent microsomal flavoprotein (L-HPhe FP; EC unclassified). Inhibition studies using Phe homologs and HPhe analogs (alpha-amino-, alpha-carboxyl- and ring-substituted), and specific amino acid modifications, were carried out to determine the possible active site structure and catalytic mechanism of L-HPhe FP. Activity with L-HPhe was inhibited by the two higher homologs, but not by L-Phe. Methylation of the substrate alpha-amino group, or replacement of the alpha-carboxyl group with a phosphonic acid group, significantly reduced the inhibition. Ring substitutions had varying effects: single methyl substitutions had only minor effects on binding to the active site, whereas di- or tri-methyl, methoxy or halide substitutions significantly reduced inhibition. Simple amines had no significant effect on L-HPhe FP activity. Binding to the active site of the enzyme appears to require a minimum chain length, plus an aromatic ring at one end of the molecule and unmodified alpha-amino acid moiety at the other. Chemical modification of amino acids on the protein implied there was no requirement for thiol groups (-SH), Ser/Thr hydroxyl groups, or L-Arg in the active site of L-HPhe FP. However, there was evidence for the presence of essential His and Tyr residues, and the involvement of Glu or Asp residues at or near the active site. (C) Elsevier, Paris.