Journal ArticleDOI
Molecular recognition in (+)-alpha-pinene oxidation by cytochrome P450cam
Stephen Bell,Xuehui Chen,Rebecca J. Sowden,Feng Xu,Jennifer N. Williams,Luet-Lok Wong,Zihe Rao +6 more
TLDR
The results show that the protein engineering of P450(cam) for high selectivity of substrate oxidation is more difficult than achieving high substrate turnover rates because of the subtle and dynamic nature of enzyme-substrate interactions.Abstract:
Oxygenated derivatives of the monoterpene (+)-α-pinene are found in plant essential oils and used as fragrances and flavorings. (+)-α-Pinene is structurally related to (+)-camphor, the natural substrate of the heme monooxygenase cytochrome P450cam from Pseudomonas putida. The aim of the present work was to apply the current understanding of P450 substrate binding and catalysis to engineer P450cam for the selective oxidation of (+)-α-pinene. Consideration of the structures of (+)-camphor and (+)-α-pinene lead to active-site mutants containing combinations of the Y96F, F87A, F87L, F87W, and V247L mutations. All mutants showed greatly enhanced binding and rate of oxidation of (+)-α-pinene. Some mutants had tighter (+)-α-pinene binding than camphor binding by the wild-type. The most active was the Y96F/V247L mutant, with a (+)-α-pinene oxidation rate of 270 nmol (nmol of P450cam)-1 min-1, which was 70% of the rate of camphor oxidation by wild-type P450cam. Camphor is oxidized by wild-type P450cam exclusively ...read more
Citations
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Journal ArticleDOI
Structure and Chemistry of Cytochrome P450
TL;DR: This review will concentrate on findings with P-450cam of the Pseudomonas putida camphor-5-exo-hydroxylase, and attention will be drawn to parallel and contrasting examples from other P- 450s as appropriate.
Journal ArticleDOI
Cytochromes P450 as versatile biocatalysts.
TL;DR: The set of interesting reactions being catalysed by cytochromes P450 systems and the availability of new genetic engineering techniques allowing to heterologously express them and to improve and change their activity, stability and selectivity makes them promising candidates for biotechnological application in the future.
Journal ArticleDOI
Biosynthesis of Plant Isoprenoids: Perspectives for Microbial Engineering
James Kirby,Jay D. Keasling +1 more
TL;DR: Investigations into isoprenoid function and gene discovery in plants as well as the latest advances in isOPrenoid pathway engineering in both plant and microbial hosts are reviewed.
Journal ArticleDOI
Enzyme-mediated oxidations for the chemist
TL;DR: The present state of the art in the use of enzymes and microorganisms for catalytic oxidation and oxyfunctionalization chemistry is reviewed.
Journal ArticleDOI
Tuning P450 Enzymes as Oxidation Catalysts
TL;DR: The development of catalytic systems for the controlled oxidation of C–H bonds remains a highly sought-after goal in chemistry owing to the great utility of such transformation toward expediting the synthesis and functionalization of organic molecules.
References
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Journal ArticleDOI
P450 superfamily: Update on new sequences, gene mapping, accession numbers and nomenclature
David R. Nelson,Luc Koymans,Tetsuya Kamataki,John J. Stegeman,Ren Feyereisen,David J. Waxman,Michael R. Waterman,Osamu Gotoh,Minor J. Coon,Ron W. Estabrook,Irwin C. Gunsalus,Daniel W. Nebert +11 more
TL;DR: This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the P450 superfamily has been described and is similar to that proposed in the previous updates.
Journal ArticleDOI
High-resolution crystal structure of cytochrome P450cam.
TL;DR: The crystal structure of Pseudomonas putida cytochrome P450 with its substrate, camphor, bound has been refined to R = 0.19 at a normal resolution of 1.63 A as discussed by the authors.
Journal ArticleDOI
The Catalytic Pathway of Cytochrome P450Cam at Atomic Resolution
Ilme Schlichting,Joel Berendzen,Kelvin Chu,Kelvin Chu,Ann M. Stock,Shelley A. Maves,David E. Benson,Robert M. Sweet,Dagmar Ringe,Gregory A. Petsko,Stephen G. Sligar,Stephen G. Sligar +11 more
TL;DR: Structures were obtained for three intermediates in the hydroxylation reaction of camphor by P450cam with trapping techniques and cryocrystallography and reveal a network of bound water molecules that may provide the protons needed for the reaction.
Journal ArticleDOI
The 2.6-A crystal structure of Pseudomonas putida cytochrome P-450.
TL;DR: The crystal structure of Pseudomonas putida cytochrome P-450cam in the ferric, camphor bound form has been determined and partially refined to R = 0.23 at 2.6 A.
Journal ArticleDOI
Crystal structure of substrate-free Pseudomonas putida cytochrome P-450.
TL;DR: Analysis of the crystal structure of Pseudomonas putida cytochrome P-450cam in the substrate-free form and a quantitative comparison of the two refined structures reveal that no detectable conformational change results from camphor binding other than a small repositioning of a phenylalanine side chain that contacts the camphor molecule.