Complete pyridine-nucleotide-specific conversion of an NADH-dependent ferredoxin reductase.

The coenzyme specificity of enzymes is one of the critical parameters for the engineered production of biological compounds using bacteria. Since NADPH is produced abundantly in photosynthetic organisms, conversion of an NADH-specific enzyme into an NADPH-specific one is a useful approach for the efficient carbon-neutral production of biological compounds in photosynthetic organisms. In the present study, an NADH-specific ferredoxin reductase component, BphA4 of biphenyl dioxygenase BphA from Acidovorax sp. strain KKS102, was changed to an NADPH-dependent form using a method combining structure-based systematic mutations and site-directed random mutagenesis. The resultant CRG mutant, in which Glu175-Thr176-Gln177 of an NADH-recognition loop in the wild-type BphA4 was replaced with Cys175-Arg176-Gly177, was highly specific and active for NADPH, and its biochemical and structural properties for NADPH were nearly the same as those of the wild-type BphA4 for NADH. In addition, this mutation project was assessed by a semi-empirical prediction method of mutation effects, and the results suggested that the CRG mutant was one of the best NADPH-specific mutants.

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.

Crystallization and preliminary X-ray analysis of the electron-transfer complex of Rieske-type [2Fe-2S] ferredoxin and NADH-dependent ferredoxin reductase derived from Acidovorax sp. strain KKS102.

The electron-transfer complex of BphA3, a Rieske-type [2Fe-2S] ferredoxin, and BphA4, a NADH-dependent ferredoxin reductase, was crystallized using the sitting-drop vapour-diffusion method under anaerobic conditions. The obtained crystals were analyzed by SDS-PAGE, which showed that they contained both BphA3 and BphA4. The crystals belong to space group P2(1), with unit-cell parameters a = 60.60, b = 173.72, c = 60.98 A, beta = 115.8 degrees, and diffracted to a resolution of 1.9 A.

Crystallization and preliminary X-ray analysis of the reduced Rieske-type [2Fe-2S] ferredoxin derived from Pseudomonas sp. strain KKS102.

The reduced form of BphA3, a Rieske-type [2Fe-2S] ferredoxin component of the biphenyl dioxygenase BphA from Pseudomonas sp. strain KKS102, was crystallized by the sitting-drop vapour-diffusion method under anaerobic conditions. The crystal belongs to space group P3(1)21, with unit-cell parameters a = b = 49.6, c = 171.9 A, and diffracts to a resolution of 1.95 A. A molecular-replacement calculation using oxidized BphA3 as a search model yielded a satisfactory solution.

Crystallization and preliminary X-ray analysis of the Rieske-type [2Fe-2S] ferredoxin component of biphenyl dioxygenase from Pseudomonas sp. strain KKS102.

BphA3, a Rieske-type [2Fe-2S] ferredoxin component of a biphenyl dioxygenase (BphA) from Pseudomonas sp. strain KKS102, was crystallized by the hanging-drop vapour-diffusion method. Two crystal forms were obtained from the same conditions. The form I crystal belongs to space group P2(1)2(1)2, with unit-cell parameters a = 26.3, b = 144.3, c = 61.5 A, and diffracted to 2.45 A resolution. The form II crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 26.2, b = 121.3, c = 142.7 A, and diffracted to 2.8 A resolution. A molecular-replacement calculation using BphF as a search model yielded a satisfactory solution for both forms.