Assembly of non-natural electron transfer conduits in the cytochrome P450 system: a critical assessment and update of artificial redox constructs amenable to exploitation in biotechnological areas

The high plasticity of the active-site cavity of cytochromes P450, permitting reactivity toward a vast array of compounds, makes these enzymes attractive targets for biotechnological application. Escalating attention in this area is driven by remarkable progress in the rational design by DNA shuffling of self-sufficient, multi-domain P450/electron donor constructs simplifying the composition of biocatalytic systems. Moreover, versatile approaches were undertaken to supersede the well-established, NAD(P)H-steered proteinaceous redox chains by cost-effective alternative electron transfer conduits constituted of organometallic mediators or photoactivatable redox triggers. Electrochemical techniques have proven particularly useful: employing different types of carbon- and metal-based electrodes for the fabrication of biosensors, the continuing challenge was to optimize the conductive properties of these devices by creating biocompatible interfaces for transferring electrons between sensor surfaces and redox proteins. The present review provides a critical update of the most significant breakthroughs in innovative manipulation of the redox machinery, giving an impulse to exploitation of P450s in fields such as the production of fine chemicals, drug processing, medicinal diagnostics and remediation of biotopes contaminated with harmful environmental pollutants.     

Selective ϖ-1 oxidation of fatty acids by CYP147G1 from Mycobacterium marinum

Background

Cyp147G1 is one of 47 cytochrome P450 encoding genes in Mycobacterium marinum M, a pathogenic bacterium with a high degree of sequence similarity to Mycobacterium tuberculosis and Mycobacterium ulcerans. Cyp147G1 is one of only two of these cyp genes which are closely associated with a complete electron transfer system.

Purification, characterization, and directed evolution study of a vitamin D3 hydroxylase from Pseudonocardia autotrophica

Vitamin D₃ (VD₃) is a fat-soluble prohormone that plays a crucial role in bone metabolism, immunity, and control of cell proliferation and cell differentiation in mammals. The actinomycete Pseudonocardia autotrophica is capable of bioconversion of VD₃ into its physiologically active forms, namely, 25(OH)VD₃ or 1α,25(OH)₂VD₃. In this study, we isolated and characterized Vdh (vitamin D₃ hydroxylase), which hydroxylates VD₃ from P. autotrophica NBRC 12743. The vdh gene encodes a protein containing 403 amino acids with a molecular weight of 44,368 Da. This hydroxylase was found to be homologous with the P450 belonging to CYP107 family. Vdh had the same ratio of the V_max values for VD₃ 25-hydroxylation and 25(OH)VD₃ 1α-hydroxylation, while other enzymes showed preferential regio-specific hydroxylation on VD₃. We characterized a collection of Vdh mutants obtained by random mutagenesis and obtained a Vdh-K1 mutant by the combination of four amino acid substitutions. Vdh-K1 showed one-order higher VD₃ 25-hydroxylase activity than the wild-type enzyme. Biotransformation of VD₃ into 25(OH)VD₃ was successfully accomplished with a Vdh-expressed recombinant strain of actinobacterium Rhodococcus erythropolis. Vdh may be a useful enzyme for the production of physiologically active forms of VD₃ by a single cytochrome P450.     

Molecular mechanism of the redox-dependent interaction between NADH-dependent ferredoxin reductase and Rieske-type [2Fe-2S] ferredoxin

The electron transfer system of the biphenyl dioxygenase BphA, which is derived from Acidovorax sp. (formally Pseudomonas sp.) strain KKS102, is composed of an FAD-containing NADH-ferredoxin reductase (BphA4) and a Rieske-type [2Fe-2S] ferredoxin (BphA3). Biochemical studies have suggested that the whole electron transfer process from NADH to BphA3 comprises three consecutive elementary electron-transfer reactions, in which BphA3 and BphA4 interact transiently in a redox-dependent manner. Initially, BphA4 receives two electrons from NADH. The reduced BphA4 then delivers one electron each to the [2Fe-2S] cluster of the two BphA3 molecules through redox-dependent transient interactions. The reduced BphA3 transports the electron to BphA1A2, a terminal oxygenase, to support the activation of dioxygen for biphenyl dihydroxylation. In order to elucidate the molecular mechanisms of the sequential reaction and the redox-dependent interaction between BphA3 and BphA4, we determined the crystal structures of the productive BphA3-BphA4 complex, and of free BphA3 and BphA4 in all the redox states occurring in the catalytic cycle. The crystal structures of these reaction intermediates demonstrated that each elementary electron transfer induces a series of redox-dependent conformational changes in BphA3 and BphA4, which regulate the interaction between them. In addition, the conformational changes induced by the preceding electron transfer seem to induce the next electron transfer. The interplay of electron transfer and induced conformational changes seems to be critical to the sequential electron-transfer reaction from NADH to BphA3.     

Molecular mechanism of metabolic NAD(P)H-dependent electron-transfer systems: The role of redox cofactors

NAD(P)H-dependent electron-transfer (ET) systems require three functional components: a flavin-containing NAD(P)H-dehydrogenase, one-electron carrier and metal-containing redox center. In principle, these ET systems consist of one-, two- and three-components, and the electron flux from pyridine nucleotide cofactors, NADPH or NADH to final electron acceptor follows a linear pathway: NAD(P)H?→?flavin?→?one-electron carrier?→?metal containing redox center. In each step ET is primarily controlled by one- and two-electron midpoint reduction potentials of protein-bound redox cofactors in which the redox-linked conformational changes during the catalytic cycle are required for the domain-domain interactions. These interactions play an effective ET reactions in the multi-component ET systems. The microsomal and mitochondrial cytochrome P450 (cyt P450) ET systems, nitric oxide synthase (NOS) isozymes, cytochrome b₅ (cyt b₅) ET systems and methionine synthase (MS) ET system include a combination of multi-domain, and their organizations display similarities as well as differences in their components. However, these ET systems are sharing of a similar mechanism. More recent structural information obtained by X-ray and cryo-electron microscopy (cryo-EM) analysis provides more detail for the mechanisms associated with multi-domain ET systems. Therefore, this review summarizes the roles of redox cofactors in the metabolic ET systems on the basis of one-electron redox potentials. In final Section, evolutionary aspects of NAD(P)H-dependent multi-domain ET systems will be discussed.