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Int. J. Mol. Sci. 2016, 17(12), 2067; doi:10.3390/ijms17122067

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

1
Unit of Polar Genomics, Korea Polar Research Institute, Incheon 406-840, Korea
2
Department of Polar Sciences, University of Science and Technology, Incheon 406-840, Korea
3
Department of BT-Convergent Pharmaceutical Engineering, Sunmoon University, Asansi 336-708, Korea
These authors contributed equally to this work.
*
Authors to whom correspondence should be addressed.
Academic Editor: Charles A. Collyer
Received: 17 October 2016 / Revised: 29 November 2016 / Accepted: 6 December 2016 / Published: 9 December 2016
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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Abstract

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. View Full-Text
Keywords: cytochrome P450; substrate binding assay; crystal structure; Sphingomonas sp.; X-ray crystallography cytochrome P450; substrate binding assay; crystal structure; Sphingomonas sp.; X-ray crystallography
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MDPI and ACS Style

Lee, C.W.; Yu, S.-C.; Lee, J.-H.; Park, S.-H.; Park, H.; Oh, T.-J.; Lee, J.H. Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding. Int. J. Mol. Sci. 2016, 17, 2067.

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