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A second cluster of genes encoding the E1␣, E1␤, and E2 subunits of branched-chain ␣-keto acid dehydrogenase (BCDH), bkdFGH, has been cloned and characterized from Streptomyces avermitilis, the soil microorganism which produces anthelmintic avermectins. Open reading frame 1 (ORF1) (bkdF, encoding E1␣), would encode a polypeptide of 44,394 Da (406 amino acids). The putative start codon of the incompletely sequenced ORF2 (bkdG, encoding E1␤) is located 83 bp downstream from the end of ORF1. The deduced amino acid sequence of bkdF resembled the corresponding E1␣ subunit of several prokaryotic and eukaryotic BCDH complexes. An S. avermitilis bkd mutant constructed by deletion of a genomic region comprising the 5 end of bkdF is also described. The mutant exhibited a typical Bkd ؊ phenotype: it lacked E1 BCDH activity and had lost the ability to grow on solid minimal medium containing isoleucine, leucine, and valine as sole carbon sources. Since BCDH provides an ␣-branched-chain fatty acid starter unit, either S(؉)-␣-methylbutyryl coenzyme A or isobutyryl coenzyme A, which is essential to initiate the synthesis of the avermectin polyketide backbone in S. avermitilis, the disrupted mutant cannot make the natural avermectins in a medium lacking both S(؉)-␣-methylbutyrate and isobutyrate. Supplementation with either one of these compounds restores production of the corresponding natural avermectins, while supplementation of the medium with alternative fatty acids results in the formation of novel avermectins. These results verify that the BCDH-catalyzed reaction of branched-chain amino acid catabolism constitutes a crucial step to provide fatty acid precursors for antibiotic biosynthesis in S. avermitilis.

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The reactivity of the imino acids formed in the D- or L-amino acid oxidase reaction was studied. It was found that: (1) When imino acids reacted with the alpha-amino group of glycine or other amino acids, transimination yielded derivatives less stable to hydrolysis than the parent imino acids. In contrast, when imino acids reacted with the epsilon-amino group of lysine or other primary amines, transimination yielded derivatives more stable to hydrolysis than the parent imino acids. (2) Imino acids react rapidly with hydrazine and semicarbazide, forming stable hydrazones and semicarbazones. At pH 7.7, the rate of reaction of the imino acid analogue of leucine with semicarbazide was 10(4) times greater than that of the corresponding keto acid. The reaction of imino acids with these reagents is rapid enough to permit one to follow spectrophotometrically the amino acid oxidase reaction. Imino acids also reacted with cyanide to yield stable adducts. (3) The rate of hydrolysis of the imino acid analogue of leucine was independent of pH above pH 8.5. At lower pH values, the rate of hydrolysis increased with decreasing pH. At 25 degrees C and in the absence of added amino compounds, this imino acid had a half-life of 22 s at pH 8.5. Its half-life was 9.9 s at pH 7.9.