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Accession Number DE2013-1035499
Title Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases.
Publication Date Feb 2012
Media Count 31p
Personal Author F. H. Arnold
Abstract The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical oxidants of various P450s directly using a chemical approach to generate the radical in situ. This resulted in the first report of direct methane to methanol conversion by a heme porphyrin catalyst using the soluble P450 from Mycobacterium sp, CYP153A6.
Keywords Alkanes
Catalysts
Cytochome P-450
Enzymes
Ethane
Fatty acids
Hydroxylanes
Kinetics
Methane
Myboacterium
Proteins
Redox processes
Substrates


 
Source Agency Technical Information Center Oak Ridge Tennessee
NTIS Subject Category 57F - Cytology, Genetics, & Molecular Biology
57K - Microbiology
99F - Physical & Theoretical Chemistry
Corporate Author California Inst. of Tech., Pasadena. Chemical Engineering Lab.
Document Type Technical report
Title Note N/A
NTIS Issue Number 1314
Contract Number DE-FG02-06ER15762

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