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 ke y step in its biosynthesis is the oxidative decarboxylation of L-HPhe to it s aldoxime. The enzyme catalysing this reaction has been shown to be a NADP H- 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, we re carried out to determine the possible active site structure and catalyti c mechanism of L-HPhe FP. Activity with L-HPhe was inhibited by the two hig her homologs, but not by L-Phe. Methylation of the substrate alpha-amino gr oup, or replacement of the alpha-carboxyl group with a phosphonic acid grou p, significantly reduced the inhibition. Ring substitutions had varying eff ects: single methyl substitutions had only minor effects on binding to the active site, whereas di- or tri-methyl, methoxy or halide substitutions sig nificantly reduced inhibition. Simple amines had no significant effect on L -HPhe FP activity. Binding to the active site of the enzyme appears to requ ire a minimum chain length, plus an aromatic ring at one end of the molecul e and unmodified alpha-amino acid moiety at the other. Chemical modificatio n 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-HPh e 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.