Two decades have passed since the discovery in liver microsomes of a haemprotein that forms a reduced-CO complex with the absorptive maximum of the Soret at 450 nm (Klingenberg, 1958; Garfinkel, 1958) and the identification of this protein as a new cytochrome: pigment cytochrome, P-450 (Omura and Sato, 1962, 1964a). In the intervening years, the study of cytochrome P-450 dependent monoxygenases has expanded exponentially. From the first crude attempts to solubilise a P-450 (Omura and Sato, 1963, 1964b) to the determination of the primary, secondary, and tertiary structure of cytochrome P-450cam by amino acid sequencing (Haniu et al., 1982a,b) and x-ray crystallography (Poulos et al., 1984) our understanding of this unique family of proteins has been advancing on all fronts. Since, perhaps, the greatest understanding of the structure and mechanism of P-450s has come from concentrated study of P-450cam of the Pseudomonas putida camphor-5-exo-hydroxylase, this review will concentrate on findings with P-450cam; attention will be drawn to parallel and contrasting examples from other P-450s as appropriate.

Structure and Chemistry of Cytochrome P450

Cytochromes P450 as versatile biocatalysts.

Isoprenoids are a large and highly diverse group of natural products with many functions in plant primary and secondary metabolism. Isoprenoids are synthesized from common prenyl diphosphate precursors through the action of terpene synthases and terpene-modifying enzymes such as cytochrome P450 monooxygenases. Many isoprenoids have important applications in areas such as human health and nutrition, and much effort has been directed toward their production in microbial hosts. However, many hurdles must be overcome in the elucidation and functional microbial expression of the genes responsible for biosynthesis of an isoprenoid of interest. Here, we review 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.

Biosynthesis of Plant Isoprenoids: Perspectives for Microbial Engineering

Enzyme-mediated oxidations for the chemist

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. Cytochrome P450 monooxygenases are the catalysts of choice in the biological world for mediating the oxidation of sp3 and sp2 C–H bonds with a high degree of chemo-, regio-, and stereoselectivity and in a wide array of compounds of varying complexity. The efficiency of these enzymes, compared with chemical methods, to catalyze the insertion of oxygen into unactivated C–H bonds under mild reaction conditions has sparked interest among researchers toward investigating and exploiting P450s for a variety of synthetic applications. Realizing the synthetic potential of these enzymes, however, depends upon the availability of effective strategies to tune the reactivity of natural P450s to obtain viable oxidation catalysts for the desired transformation. This revie...

Tuning P450 Enzymes as Oxidation Catalysts

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 ...

Feng Xu

Papers

Molecular recognition in (+)-alpha-pinene oxidation by cytochrome P450cam