title: Mechanistic Study of the Stereoselective Hydroxylation of [2-2H1,3-2H1]Butanes Catalyzed by Cytochrome P450 BM3 Variants.
abstract: Engineered bacterial cytochrome P450s are noted for their ability in the oxidation of inert small alkanes. Cytochrome P450 BM3 L188P A328F (BM3 PF) and A74E L188P A328F (BM3 EPF) variants are able to efficiently oxidize n-butane to 2-butanol. Esterification of the 2-butanol derived from this reaction mediated by the aforementioned two mutants gives diastereomeric excesses (de) of -56±1 and -52±1 %, respectively, with the preference for the oxidation occurring at the C-HSbond. When tailored (2R,3R)- and (2S,3S)-[2-2H1,3-2H1]butane probes are employed as substrates for both variants, the obtained de values from (2R,3R)-[2-2H1,3-2H1]butane are -93 and -92 % for BM3 PF and EPF, respectively; whereas the obtained de values from (2S,3S)-[2-2H1,3-2H1]butane are 52 and 56 % in the BM3 PF and EPF systems, respectively. The kinetic isotope effects (KIEs) for the oxidation of (2R,3R)-[2-2H1,3-2H1]butane are 7.3 and 7.8 in BM3 PF and EPF, respectively; whereas KIEs for (2S,3S)-[2-2H1,3-2H1]butanes are 18 and 25 in BM3 PF and EPF, respectively. The discrepancy in KIEs obtained from the two substrates supports the two-state reactivity (TSR) that is proposed for alkane oxidation in cytochrome P450 systems. Moreover, for the first time, experimental evidence for tunneling in the oxidation mediated by P450 is given through the oxidation of the C-HRbond in (2S,3S)-[2-2H1,3-2H1]butane.
authors: Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan.|Graduate Institute of Applied Science and Technology, National (Taiwan) University of Science and Technology, Taipei, 106, Taiwan.
authors: Chung-Ling
authors: CL
authors: Yang
authors: Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan.|Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi, 621, Taiwan.
authors: Cheng-Hung
authors: CH
authors: Lin
authors: Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan.
authors: Wen-I
authors: WI
authors: Luo
authors: Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan.|Graduate Institute of Applied Science and Technology, National (Taiwan) University of Science and Technology, Taipei, 106, Taiwan.
authors: Tsu-Lin
authors: TL
authors: Lee
authors: Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan.
authors: Ravirala
authors: R
authors: Ramu
authors: Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan.
authors: Kok Yaoh
authors: KY
authors: Ng
authors: Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan.
authors: Yi-Fang
authors: YF
authors: Tsai
authors: Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi, 621, Taiwan.
authors: Guor-Tzo
authors: GT
authors: Wei
authors: Institute of Chemistry, Academia Sinica, Taipei, 115, Taiwan.|Graduate Institute of Applied Science and Technology, National (Taiwan) University of Science and Technology, Taipei, 106, Taiwan.
authors: Steve S-F
authors: http://orcid.org/0000-0002-3462-065X
authors: SS
authors: Yu
mesh_terms: D001419:Bacteria / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D002073:Butanes / Q000737:chemistry / Q000378:metabolism*; D000440:Butanols / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D020134:Catalytic Domain; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D006900:Hydroxylation; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D013237:Stereoisomerism; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D002073:Butanes; D000440:Butanols; C043958:2-butanol; C046888:butane; D003577:Cytochrome P-450 Enzyme System
keywords: cytochrome P450; enzyme catalysis; heme proteins; isotope effects; oxidation; stereoselectivity