title: Characterisation of two self-sufficient CYP102 family monooxygenases from Ktedonobacter racemifer DSM44963 which have new fatty acid alcohol product profiles.
abstract: BACKGROUND
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Chemistry, University of Adelaide, SA 5005, Australia.
authors.forename: Samuel D
authors.initials: SD
authors.lastname: Munday
authors.affiliation: Department of Chemistry, University of Adelaide, SA 5005, Australia.
authors.forename: Natasha K
authors.initials: NK
authors.lastname: Maddigan
authors.affiliation: Department of Chemistry, University of Adelaide, SA 5005, Australia.
authors.forename: Rosemary J
authors.initials: RJ
authors.lastname: Young
authors.affiliation: Department of Chemistry, University of Adelaide, SA 5005, Australia. Electronic address: stephen.bell@adelaide.edu.au.
authors.forename: Stephen G
authors.initials: SG
authors.lastname: Bell
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D041862:Chloroflexi / Q000201:enzymology*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry*; D005227:Fatty Acids / Q000737:chemistry*; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry*; D010802:Phylogeny
chemical_list: D001426:Bacterial Proteins; D005227:Fatty Acids; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases
keywords: Chloroflexi; Cytochrome P450 enzymes; Fatty acid oxidation; Ktedonobacter; Monooxygenases