title: Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding.
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 bacteriumSphingomonassp. PAMC 26605. Substrate affinities to C10-C12 n-alkanes and C10-C14 fatty acids withKdvalues 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.
authors.affiliation: Unit of Polar Genomics, Korea Polar Research Institute, Incheon 406-840, Korea. justay@kopri.re.kr.|Department of Polar Sciences, University of Science and Technology, Incheon 406-840, Korea. justay@kopri.re.kr.
authors.forename: Chang Woo
authors.initials: CW
authors.lastname: Lee
authors.affiliation: Department of BT-Convergent Pharmaceutical Engineering, Sunmoon University, Asansi 336-708, Korea. ysc9056@naver.com.
authors.forename: Sang-Cheol
authors.initials: SC
authors.lastname: Yu
authors.affiliation: Department of BT-Convergent Pharmaceutical Engineering, Sunmoon University, Asansi 336-708, Korea. shadowjhl@empas.com.
authors.forename: Joo-Ho
authors.initials: JH
authors.lastname: Lee
authors.affiliation: Unit of Polar Genomics, Korea Polar Research Institute, Incheon 406-840, Korea. psh@kopri.re.kr.
authors.forename: Sun-Ha
authors.initials: SH
authors.lastname: Park
authors.affiliation: Unit of Polar Genomics, Korea Polar Research Institute, Incheon 406-840, Korea. hpark@kopri.re.kr.|Department of Polar Sciences, University of Science and Technology, Incheon 406-840, Korea. hpark@kopri.re.kr.
authors.forename: Hyun
authors.initials: H
authors.lastname: Park
authors.affiliation: Department of BT-Convergent Pharmaceutical Engineering, Sunmoon University, Asansi 336-708, Korea. tjoh3782@sunmoon.ac.kr.
authors.forename: Tae-Jin
authors.initials: TJ
authors.lastname: Oh
authors.affiliation: Unit of Polar Genomics, Korea Polar Research Institute, Incheon 406-840, Korea. junhyucklee@kopri.re.kr.|Department of Polar Sciences, University of Science and Technology, Incheon 406-840, Korea. junhyucklee@kopri.re.kr.
authors.forename: Jun Hyuck
authors.initials: JH
authors.lastname: Lee
mesh_terms: D018360:Crystallography, X-Ray; D042926:Cytochrome P-450 CYP4A / Q000737:chemistry* / Q000378:metabolism*; D008968:Molecular Conformation; D011485:Protein Binding; D020578:Sphingomonas / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D042926:Cytochrome P-450 CYP4A
keywords: Sphingomonas sp.; X-ray crystallography; crystal structure; cytochrome P450; substrate binding assay