Characterisation of two self-sufficient CYP102 family monooxygenases from Ktedonobacter racemifer DSM44963 which have new fatty acid alcohol product profiles

• Published in: Biochimica et biophysica acta Vol. 1860; no. 6; pp. 1149 - 1162
• Main Authors: Munday, Samuel D., Maddigan, Natasha K., Young, Rosemary J., Bell, Stephen G.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.06.2016
• Subjects: alcohols; Amino Acid Sequence; bacteria; Bacterial Proteins - chemistry; Catalytic Domain; Chloroflexi; Chloroflexi - enzymology; cytochrome P-450; Cytochrome P-450 Enzyme System - chemistry; Cytochrome P450 enzymes; enzymes; Fatty acid oxidation; Fatty Acids - chemistry; genes; in vitro studies; Ktedonobacter; Molecular Sequence Data; Monooxygenases; NADP (coenzyme); NADPH-Ferrihemoprotein Reductase - chemistry; oxidation; Phylogeny; saturated fatty acids; unsaturated fatty acids; NADPH; TMSCl; Fatty acid oxidation; Cytochrome P450 enzymes; EMM; DMSO; MS; EtOH; Ktedonobacter; Tris; NADH; Monooxygenases; BSTFA; IPTG; 2xYT; Chloroflexi; DEAE; CYP; GC; HPLC; PCR
• ISSN: 0304-4165; 0006-3002; 1872-8006
• DOI: 10.1016/j.bbagen.2016.01.023

Two self-sufficient CYP102 family encoding genes (Krac_0936 and Krac_9955) from the bacterium Ktedonobacter racemifer DSM44963, which possesses one of the largest bacterial genomes, have been identified. Phylogenetic analysis of both the encoded cytochrome P450 enzymes, Krac0936 and Krac9955. Both enzymes were produced and their turnovers with fatty acid substrates assessed in vitro and using a whole-cell oxidation system. Krac0936 hydroxylated straight chain, saturated fatty acids predominantly at the ω-1 and ω-2 positions using NADPH as the cofactor. Krac0936 was less active towards shorter unsaturated fatty acids but longer unsaturated acids were efficiently oxidised. cis,cis-9,12-Octadecadienoic and pentadecanoic acids were the most active substrates tested with Krac0936. Unusually Krac9955 showed very low levels of NAD(P)H oxidation activity though coupling of the reducing equivalents to product formation was high. The product distribution of tridecanoic, tetradecanoic and pentadecanoic acid oxidation by Krac9955 favoured oxidation at the ω-4, ω-5 and ω-6 positions, respectively. Krac0936 and Krac9955 are self-sufficient P450 monooxygenases. Krac0936 has a preference for pentadecanoic acid over other straight chain fatty acids and showed little or no activity with dodecanoic or octadecanoic acids. Krac9955 preferably oxidised shorter fatty acids compared to Krac0936 with tridecanoic having the highest levels of product formation. Unlike Krac0936 and P450Bm3, Krac9995 showed lower activities with unsaturated fatty acids. In this study of two of the CYP enzymes from K. racemifer we have shown that this bacterium from the Chloroflexi phylum contains genes which encode new proteins with novel activity. •Identification of two self-sufficient cytochrome P450 genes in K. racemifer DSM44963•Analysis of the relationship of Krac0936 and Krac9955 with other P450Bm3 like enzymes•Purification and preliminary characterisation of both enzymes•The observation of modified selectivity of fatty acid oxidation by Krac0936 compared to P450Bm3•Krac9955 has an unusual substrate range and product selectivity.