TY - JOUR
T1 - Characterisation of two self-sufficient CYP102 family monooxygenases from Ktedonobacter racemifer DSM44963 which have new fatty acid alcohol product profiles
AU - Munday, Samuel D.
AU - Maddigan, Natasha K.
AU - Young, Rosemary J.
AU - Bell, Stephen G.
N1 - Funding Information:
The authors thank the University of Adelaide for the award of a M. Phil Scholarship (to SDM). SGB thanks the Australian Research Council for a Future Fellowship (FT FT140100355 ).
Publisher Copyright:
© 2016 Elsevier B.V. All rights reserved.
Copyright:
Copyright 2017 Elsevier B.V., All rights reserved.
PY - 2016/6/1
Y1 - 2016/6/1
N2 - 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. Conclusion: 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. General significance: 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.
AB - 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. Conclusion: 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. General significance: 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.
KW - Chloroflexi
KW - Cytochrome P450 enzymes
KW - Fatty acid oxidation
KW - Ktedonobacter
KW - Monooxygenases
UR - http://www.scopus.com/inward/record.url?scp=84960194071&partnerID=8YFLogxK
U2 - 10.1016/j.bbagen.2016.01.023
DO - 10.1016/j.bbagen.2016.01.023
M3 - Article
C2 - 26825771
AN - SCOPUS:84960194071
SN - 0304-4165
VL - 1860
SP - 1149
EP - 1162
JO - Biochimica et Biophysica Acta. General Subjects
JF - Biochimica et Biophysica Acta. General Subjects
IS - 6
ER -