Genes encoding acyl-CoA dehydrogenase (AcdH) homologues from Streptomyces coelicolor and Streptomyces avermitilis provide insights into the metabolism of small branched-chain fatty acids and macrolide antibiotic production

School of Pharmacy 1 and Department of Bacteriology 2 , University of Wisconsin, 425 N. Charter St, Madison, WI 53706, USA Bioprocess Research, Central Research Division, Pfizer Inc., Groton, CT 06340, USA 3 Department of Medicinal Chemistry, School of Pharmacy and Institute for Structural Biology and Drug Discovery, Virginia Commonwealth University, Richmond, VA 23219, USA 4 Author for correspondence: C. Richard Hutchinson. Tel: +1 608 262 7582. Fax: +1 608 262 3134. e-mail: crhutchi{at}facstaff.wisc.edu The cloning, using a PCR approach, of genes from both Streptomyces coelicolor and Streptomyces avermitilis encoding an acyl-CoA dehydrogenase (AcdH), putatively involved in the catabolism of branched-chain amino acids, is reported. The deduced amino acid sequences of both genes have a high similarity to prokaryotic and eukaryotic short-chain acyl-CoA dehydrogenases. When the S. coelicolor and S. avermitilis acyl-CoA dehydrogenase genes ( acdH ) were expressed in Escherichia coli , each of the AcdH flavoproteins was able to oxidize the branched-chain acyl-CoA derivatives isobutyryl-CoA, isovaleryl-CoA and cyclohexylcarbonyl-CoA, as well as the short straight-chain acyl-CoAs n -butyryl-CoA and n -valeryl-CoA in vitro . NMR spectral data confirmed that the oxidized product of isobutyryl-CoA is methacrylyl-CoA, which is the expected product at the acyl-CoA dehydrogenase step in the catabolism of valine in streptomycetes. Disruption of the S. avermitilis acdH produced a mutant unable to grow on solid minimal medium containing valine, isoleucine or leucine as sole carbon sources. Feeding studies with 13 C triple-labelled isobutyrate revealed a significant decrease in the incorporation of label into the methylmalonyl-CoA-derived positions of avermectin in the acdH mutant. In contrast the mutation did not affect incorporation into the malonyl-CoA-derived positions of avermectin. These results are consistent with the acdH gene encoding an acyl-CoA dehydrogenase with a broad substrate specificity that has a role in the catabolism of branched-chain amino acids in S. coelicolor and S. avermitilis . Keywords: avermectin, branched-chain amino acids, isotope labelling Abbreviations: AcdH, the specific acyl-CoA dehydrogenases produced by S. avermitilis and S. coelicolor ; AD, any other acyl-CoA dehydrogenase; PMS, phenazine methosulfate The GenBank accession numbers for the sequences described in this paper are AF142581 ( Streptomyces coelicolor ) and AF143210 ( Streptom