Hydroxylation of long chain fatty acids by CYP147F1, a new cytochrome P450 subfamily protein from Streptomyces peucetius

Bhattarai, Saurabh; Liou, Kwangkyoung; Oh, Tae‐Jin

doi:10.1016/j.abb.2013.09.008

Cited by 29 publications

(21 citation statements)

References 38 publications

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“…Sequence analysis revealed that CYP153D17 has 48.27% identity with P450pyr hydroxylase from Sphingopyxis macrogoltabida and that it contains conserved motifs including an oxygen-binding domain and heme-binding domains found in typical CYPs [20]. When over-expressed heterologously in Escherichia coli C41 (DE3), as previously reported by our group [21], high levels of folded CYP153D17 were obtained.…”

Section: Resultsmentioning

confidence: 79%

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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Section: Resultsmentioning

confidence: 79%

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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“…The in vitro reconstituted system was supported by redox partners PDX ( camA ) and PDR ( camB ) . They were expressed as His‐tagged proteins in E. coli BL21(DE3) by use of plasmid constructs pET28a(+) and pET32a(+) as described previously . For the in vivo system, the camA ‐harboring plasmid pET28a_PDX was digested with NdeI and XhoI restriction enzymes to obtain camA .…”

Section: Methodsmentioning

confidence: 99%

Tracking Down a New Steroid‐Hydroxylating Promiscuous Cytochrome P450: CYP154C8 from Streptomyces sp. W2233‐SM

et al. 2018

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CYP154C8 from Streptomyces sp. has been identified as a new cytochrome P450 with substrate flexibility towards different sets of steroids. In vitro treatment of these steroids with CYP154C8 revealed interesting product formation patterns with the same group of steroids. NMR study revealed the major product of corticosterone to be hydroxylated at the C21 position, whereas progesterone, androstenedione, testosterone, and 11-ketoprogesterone were exclusively hydroxylated at the 16α position. However, the 16α-hydroxylated product of progesterone was further hydroxylated to yield dihydroxylated products. 16-hydroxyprogesterone was hydroxylated at two positions to yield dihydroxylated products: 2α,16α-dihydroxyprogesterone and 6β,16α-dihydroxyprogesterone. To the best of our knowledge, this is the first report of generation of such products through enzymatic hydroxylation by a CYP450. In view of the importance of modified steroids as pharmaceutical components, CYP154C8 has immense potential for utilization in bioproduction of hydroxylated derivative compounds to be directly employed for pharmaceutical applications.

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“…Enzymatic in vitro assay with NADPH and its surrogate redox partners Fdx and Fdr : The heterologous expression and purification of CYP154C8 was performed as described previously . Similarly, the surrogate redox partners Pdx and Pdr were expressed and purified as described elsewhere . Reaction mixtures contained substrate (3 μ m ), CYP154C8 (3 μ m ), Fdx (6 μ m ), Fdr (0.1 U), glucose 6‐phosphate (10 m m ), glucose 6‐phosphate dehydrogenase (1 U), catalase (100 μg mL −1 ), MgCl 2 (1 m m ), and NADPH (250 μ m ).…”

Section: Methodsmentioning

confidence: 99%

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

Dangi

Park

2018

ChemBioChem

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CYP154C8 catalyzes the hydroxylation of diverse steroids, as has previously been demonstrated, by using an NADH-dependent system including putidaredoxin and putidaredoxin reductase as redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8 reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase displayed catalytic activity different from that of the NADH-dependent system. The NADPH-dependent system showed multistep oxidation of progesterone and other substrates including androstenedione, testosterone, and nandrolone. (Diacetoxyiodo)benzene was employed to generate compound I (FeO ), actively supporting the redox reactions catalyzed by CYP154C8. In addition to 16α-hydroxylation, progesterone and 11-oxoprogesterone also underwent hydroxylation at the 6β-position in reactions supported by (diacetoxyiodo)benzene. CYP154C8 was active in the presence of high concentrations (>10 mm) of H O , with optimum conversion surprisingly being achieved at ≈75 mm H O . More importantly, H O tolerance by CYP154C8 was evident in the very low heme oxidation rate constant (K) even at high concentrations of H O . Our results demonstrate that alternative redox partners and oxidizing agents influence the catalytic efficiency and product distribution of a cytochrome P450 enzyme. More importantly, these choices affected the type and selectivity of reaction catalyzed by the P450 enzyme.

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Hydroxylation of long chain fatty acids by CYP147F1, a new cytochrome P450 subfamily protein from Streptomyces peucetius

Cited by 29 publications

References 38 publications

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

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

Tracking Down a New Steroid‐Hydroxylating Promiscuous Cytochrome P450: CYP154C8 from Streptomyces sp. W2233‐SM

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

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