Kinetic and structural insight into a role of the re-face Tyr328 residue of the homodimer type ferredoxin-NADP+ oxidoreductase from Rhodopseudomonas palustris in the reaction with NADP+/NADPH

金沢大学理工研究域物質化学系Among the thioredoxin reductase-type ferredoxin-NAD(P)+ oxidoreductase (FNR) family, FNR from photosynthetic purple non‑sulfur bacterium Rhodopseudomonas palustris (RpFNR) is distinctive because the predicted residue on the re-face of the isoalloxazine ring portion of the FAD prosthetic group is a tyrosine. Here, we report the crystal structure of wild type RpFNR and kinetic analyses of the reaction of wild type, and Y328F, Y328H and Y328S mutants with NADP+/NADPH using steady state and pre-steady state kinetic approaches. The obtained crystal structure of wild type RpFNR confirmed the presence of Tyr328 on the re-face of the isoalloxazine ring of the FAD prosthetic group through the unique hydrogen bonding of its hydroxyl group. In the steady state assays, the substitution results in the decrease of Kd for NADP+ and KM for NADPH in the diaphorase assay; however, the kcat values also decreased significantly. In the stopped-flow spectrophotometry, mixing oxidized RpFNRs with NADPH and reduced RpFNRs with NADP+ resulted in rapid charge transfer complex formation followed by hydride transfer. The observed rate constants for the hydride transfer in both directions were comparable (>400 s−1). The substitution did not drastically affect the rate of hydride transfer, but substantially slowed down the subsequent release and re-association of NADP+/NADPH in both directions. The obtained results suggest that Tyr328 stabilizes the stacking of C-terminal residues on the isoalloxazine ring portion of the FAD prosthetic group, which impedes the access of NADP+/NADPH on the isoalloxazine ring portions, in turn, enhancing the release of the NADP+/NADPH and/or reaction with electron transfer proteins. © 2019 Elsevier B.V.Embargo Period 12 month

Crystallization and preliminary X-ray studies of ferredoxin-NAD(P)+ reductase from Chlorobium tepidum

Ferredoxin-NAD(P)+ reductase from C. tepidum has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.4 Å resolution

Structural Characterization of Heme Environmental Mutants of CgHmuT that Shuttles Heme Molecules to Heme Transporters

Corynebacteria contain a heme uptake system encoded in hmuTUV genes, in which HmuT protein acts as a heme binding protein to transport heme to the cognate transporter HmuUV. The crystal structure of HmuT from Corynebacterium glutamicum (CgHmuT) reveals that heme is accommodated in the central cleft with His141 and Tyr240 as the axial ligands and that Tyr240 forms a hydrogen bond with Arg242. In this work, the crystal structures of H141A, Y240A, and R242A mutants were determined to understand the role of these residues for the heme binding of CgHmuT. Overall and heme environmental structures of these mutants were similar to those of the wild type, suggesting that there is little conformational change in the heme-binding cleft during heme transport reaction with binding and the dissociation of heme. A loss of one axial ligand or the hydrogen bonding interaction with Tyr240 resulted in an increase in the redox potential of the heme for CgHmuT to be reduced by dithionite, though the wild type was not reduced under physiological conditions. These results suggest that the heme environmental structure stabilizes the ferric heme binding in CgHmuT, which will be responsible for efficient heme uptake under aerobic conditions where Corynebacteria grow

Crystal structural analysis of aldoxime dehydratase from Bacillus sp. OxB-1: Importance of surface residues in optimization for crystallization

Matsui D, Muraki N, Chen K, et al. Crystal structural analysis of aldoxime dehydratase from Bacillus sp. OxB-1: Importance of surface residues in optimization for crystallization. Journal of Inorganic Biochemistry . 2022;230: 111770.Aldoxime dehydratase (Oxd) is a heme enzyme that catalyzes aldoxime dehydration to the corresponding nitriles. Unlike many other heme enzymes, Oxd has a unique feature that the substrate binds directly to the heme. Therefore, it is thought that structural differences around the bound heme directly relate to differences in substrate selection. However sufficient structural information to discuss the substrate specificity has not been obtained. Oxd from Bacillus sp. OxB-1 (OxdB) shows unique substrate specificity and enantioselectivity compared to the Oxds whose crystal structures have already been reported. Here, we report the crystal structure of OxdB, which has not been reported previously. Although the crystallization of OxdB has been difficult, by adding a site-specific mutation to Glu85 located on the surface of the protein, we succeeded in crystallizing OxdB without reducing the enzyme activity. The catalytic triad essential for Oxd activity were structurally conserved in OxdB. In addition, the crystal structure of the Michaelis complex of OxdB and the diastereomerically pure substrate Z-2-(3-bromophenyl)-propanal oxime implied the importance of several hydrophobic residues for substrate specificity. Mutational analysis implicated Ala12 and Ala14 in the E/Z selectivity of bulky compounds. The N-terminal region of OxdB was shown to be shorter than those of Oxds from Pseudomonas chlororaphis and Rhodococcus sp. N-771, and have high flexibility. These structural differences possibly result in distinct preferences for aldoxime substrates based on factors such as substrate size. Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved

Raw diffraction images of 4-Methoxybenzamidine-bound trypsin

Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. We used datasets to investigate the protocol of detecting polymorphs using Hierarchical clustering (Acta D., submitted). All diffraction data were collected at BL32XU, SPring-8, using an automated data collection system ZOO. Data were acquired from four crystals of 4-Methoxybenzamidine -bound trypsin. All datasets were collected using a continuous helical scan scheme for 360º oscillation with the following experimental parameters; Beam size: 10 µm × 15 µm, Wavelength: 1.0000 Å, Total dose/crystal: 10 MGy, Detector: EIGER X 9M (DECTRIS Co. Ltd.). All crystals belonged to space group P212121 with unit cell parameters roughly corresponding to a=54.6, b=58.6, c=66.7 Å

Raw diffraction images of [NiFe]-hydrogenase maturation factor HypD from Aquifex aeolicus (C360S mutant)

HypD is one of the maturation factors of [NiFe]-hydrogenase and can form a complex with other maturation factors (Muraki et al., 2019). We used datasets to investigate the protocol of detecting polymorphs using Hierarchical clustering (Acta D., submitted). All diffraction datasets were collected at BL45XU, SPring-8, using an automated data collection system ZOO. From six crystals, datasets were collected from each one using a continuous helical scan scheme for 360º oscillation with the following experimental parameters; Beam size: 20 µm × 20 µm, Wavelength: 1.0000 Å, Total dose/crystal: 10 MGy, Detector: EIGER X 9M (DECTRIS Co. Ltd.). All crystals belonged to space group P212121 with unit cell parameters roughly corresponding to a=60.0, b=62.6, c=97.6 Å

Raw diffraction images of 5-Chlorotryptamine-bound trypsin

Trypsin is an enzyme in the first section of the small intestine that starts the digestion of protein molecules by cutting these long chains of amino acids into smaller pieces. We used datasets to investigate the protocol of detecting polymorphs using Hierarchical clustering (Acta D., submitted). All diffraction data were collected at BL32XU, SPring-8, using an automated data collection system ZOO. Data were acquired from four crystals of 5-Chlorotryptamine-bound trypsin. All datasets were collected using a continuous helical scan scheme for 360º oscillation with the following experimental parameters; Beam size: 10 µm × 15 µm, Wavelength: 1.0000 Å, Total dose/crystal: 10 MGy, Detector: EIGER X 9M (DECTRIS Co. Ltd.). All crystals belonged to space group P212121 with unit cell parameters roughly corresponding to a=54.5, b=58.6, c=66.6 Å