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

Munday, Samuel D.; Maddigan, Natasha K.; Young, Rosemary J.; Bell, Stephen

doi:10.1016/j.bbagen.2016.01.023

Cited by 25 publications

(22 citation statements)

References 59 publications

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“…The cytochrome P450 CYP102A1 (P450Bm3) from Bacillus megaterium oxidises fatty acid substrates close to the omega terminus at high activities [27,28]. It and other members of the CYP102A subfamily are unusual in that the electron transfer partner domain is fused to the heme domain [29][30][31][32]. CYP102A1 is soluble, easy to produce and the self-sufficient nature and high activity overcome two of the major hurdles to the use of P450 enzymes in synthesis.…”

Section: Introductionmentioning

confidence: 99%

Stereoselective hydroxylation of isophorone by variants of the cytochromes P450 CYP102A1 and CYP101A1

Dezvarei

Lee

Bell

2018

Enzyme and Microbial Technology

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The stereoselective oxidation of hydrocarbons is an area of research where enzyme biocatalysis can make a substantial impact. The cyclic ketone isophorone was stereoselectively hydroxylated (≥95%) by wild-type CYP102A1 to form (R)-4-hydroxyisophorone, an important chiral synthon and flavour and fragrance compound. CYP102A1 variants were also selective for 4-hydroxyisophorone formation and the product formation rate increased over the wild-type enzyme by up to 285-fold, with the best mutants being R47L/Y51F/I401P and A74G/F87V/L188Q. The latter variant, which contained mutations in the distal substrate binding pocket, was marginally less selective. Combining perfluorodecanoic acid decoy molecules with the rate accelerating variant R47L/Y51F/I401P engendered further improvement with the purified enzymes. However when the decoy molecules were used with A74G/F87V/L188Q the amount of product generated by the enzyme was reduced. Addition of decoy molecules to whole-cell turnovers did not improve the productivity of these CYP102A1 systems. WT CYP101A1 formed significant levels of 7-hydroxyisophorone as a minor product alongside 4-hydroxyisophorone. However the F87W/Y96F/L244A/V247L CYP101A1 mutant was ≥98% selective for (R)-4-hydroxyisophorone. A comparison of the two enzyme systems using whole-cell oxidation reactions showed that the best CYP101A1 variant was able to generate more product. We also characterised that the further oxidation metabolite 4-ketoisophorone was produced and then subsequently reduced to levodione by an endogenous Escherichia coli ene reductase.

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Section: Introductionmentioning

confidence: 99%

Stereoselective hydroxylation of isophorone by variants of the cytochromes P450 CYP102A1 and CYP101A1

Dezvarei

Lee

Bell

2018

Enzyme and Microbial Technology

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“…In addition, researchers have attempted to engineer enzymes from the CYP101 and CYP102 families for alkane hydroxylation. For example, P450 BM-3 from Bacillus megaterium , a well-characterized CYP102 enzyme that functions as a fatty acid monooxygenase, has been used for this purpose [14]. To create useful oxidation biocatalysts for alkane compounds, P450 BM-3 enzyme was engineered to hydroxylate linear alkanes by directed evolution with mutagenesis [15].…”

Section: Introductionmentioning

confidence: 99%

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

Lee

et al. 2016

IJMS

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Enzymatic alkane hydroxylation reactions are useful for producing pharmaceutical and agricultural chemical intermediates from hydrocarbons. Several cytochrome P450 enzymes catalyze the regio- and stereo-specific hydroxylation of alkanes. We evaluated the substrate binding of a putative CYP alkane hydroxylase (CYP153D17) from the bacterium Sphingomonas sp. PAMC 26605. Substrate affinities to C10–C12 n-alkanes and C10–C14 fatty acids with Kd values varied from 0.42 to 0.59 μM. A longer alkane (C12) bound more strongly than a shorter alkane (C10), while shorter fatty acids (C10, capric acid; C12, lauric acid) bound more strongly than a longer fatty acid (C14, myristic acid). These data displayed a broad substrate specificity of CYP153D17, hence it was named as a putative CYP alkane hydroxylase. Moreover, the crystal structure of CYP153D17 was determined at 3.1 Å resolution. This is the first study to provide structural information for the CYP153D family. Structural analysis showed that a co-purified alkane-like compound bound near the active-site heme group. The alkane-like substrate is in the hydrophobic pocket containing Thr74, Met90, Ala175, Ile240, Leu241, Val244, Leu292, Met295, and Phe393. Comparison with other CYP structures suggested that conformational changes in the β1–β2, α3–α4, and α6–α7 connecting loop are important for incorporating the long hydrophobic alkane-like substrate. These results improve the understanding of the catalytic mechanism of CYP153D17 and provide valuable information for future protein engineering studies.

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“…In contrast to this, Krac_0936, a CYP102 from the same organism, shows the "classical" pattern for saturated fatty acids but a higher activity towards oleic, linoleic, palmitoleic and myristoleic acid than Krac_9955, however, with no epoxidation (Table 6, entries 4-7, 11, and 12). Interestingly, Krac_9955 demonstrates some selectivity for allylic positions, as the terminal functionalized 10-undecenoic acid gave perfect selectivity in the allylic position and for myristoleic acid, even the in-chain allylic position was reactive (Table 6, entries 4 and 5) [74].…”

Section: Cyp102mentioning

confidence: 99%

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

2017

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

Abstract: 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.

Cited by 25 publications

References 59 publications

Stereoselective hydroxylation of isophorone by variants of the cytochromes P450 CYP102A1 and CYP101A1

Stereoselective hydroxylation of isophorone by variants of the cytochromes P450 CYP102A1 and CYP101A1

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding

Regioselective Biocatalytic Hydroxylation of Fatty Acids by Cytochrome P450s

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