Masahiko Okai

Calaxin drives sperm chemotaxis by Ca2+-mediated direct modulation of a dynein motor

Sperm chemotaxis occurs widely in animals and plants and plays an important role in the success of fertilization. Several studies have recently demonstrated that Ca2+ influx through specific Ca2+ channels is a prerequisite for sperm chemotactic movement. However, the regulator that modulates flagellar movement in response to Ca2+ is unknown. Here we show that a neuronal calcium sensor, calaxin, directly acts on outer-arm dynein and regulates specific flagellar movement during sperm chemotaxis. Calaxin inhibition resulted in significant loss of sperm chemotactic movement, despite normal increases in intracellular calcium concentration. Using a demembranated sperm model, we demonstrate that calaxin is essential for generation and propagation of Ca2+-induced asymmetric flagellar bending. An in vitro motility assay revealed that calaxin directly suppressed the velocity of microtubule sliding by outer-arm dynein at high Ca2+ concentrations. This study describes the missing link between chemoattractant-mediated Ca2+ signaling and motor-driven microtubule sliding during sperm chemotaxis.

Crystal Structure of γ-Hexachlorocyclohexane Dehydrochlorinase LinA from Sphingobium japonicum UT26

LinA from Sphingobium japonicum UT26 catalyzes two steps of dehydrochlorination from γ hexachlorocyclohexane (HCH) to 1,3,4,6-tetrachloro-1,4-cyclohexadiene via γ-pentachlorocyclohexene. We determined the crystal structure of LinA at 2.25 Å by single anomalous dispersion. LinA exists as a homotrimer, and each protomer forms a cone-shaped α+β barrel fold. The C-terminal region of LinA is extended to the neighboring subunit, unlike that of scytalone dehydratase from Magnaporthe grisea, which is one of the most structurally similar proteins identified by the DALI server. The structure we obtained in this study is in open form, in which γ-HCH can enter the active site. There is a hydrophobic cavity inside the barrel fold, and the active site is largely surrounded by the side chains of K20, L21, V24, D25, W42, L64, F68, C71, H73, V94, L96, I109, F113, and R129. H73 was considered to function as a base that abstracts the proton of γ-HCH through its interaction with D25. Docking simulations with γ-HCH and γ-pentachlorocyclohexene suggest that 11 residues (K20, I44, L64, V94, L96, I109, A111, F113, A131, C132, and T133) are involved in the binding of these compounds and support the degradation mechanism.

Crystal Structure and Site-Directed Mutagenesis Analyses of Haloalkane Dehalogenase LinB from Sphingobium sp. Strain MI1205

The enzymes LinB(UT) and LinB(MI) (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of β-hexachlorocyclohexane (β-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB(MI) and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB(MI) were categorized into three groups based on the efficiency of the first-step (from β-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB(MI) and its seven point mutants indicated how each mutated residue contributed to the first- and second-step conversions by LinB(MI). The dynamics simulation analyses of wild-type LinB(MI) and LinB(UT) revealed that the entrance of the substrate access tunnel of LinB(UT) was more flexible than that of LinB(MI), which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.

High pressure refolding, purification, and crystallization of flavin reductase from Sulfolobus tokodaii strain 7.

Flavin reductase HpaC(St) catalyzes the reduction of free flavins using NADH or NADPH. High hydrostatic pressure was used for the solubilization and refolding of HpaC(St), which was expressed as inclusion bodies in Escherichia coli to achieve high yield in a flavin-free form. The refolded HpaC(St) was purified using Ni-affinity chromatography followed by a heat treatment, which gave a single band on SDS-PAGE. The purified refolded HpaC(St) did not contain FMN, unlike the same enzyme expressed as a soluble protein. After the addition of FMN to the protein solution, the refolded enzyme showed a higher activity than the enzyme expressed as the soluble protein. Crystals of the refolded enzyme were obtained by adding FMN, FAD, or riboflavin to the protein solution and without the addition of flavin compound.

A new target region for changing the substrate specificity of amine transaminases

(R)-stereospecific amine transaminases (R-ATAs) are important biocatalysts for the production of (R)-amine compounds in a strict stereospecific manner. An improved R-ATA, ATA-117-Rd11, was successfully engineered for the manufacture of sitagliptin, a widely used therapeutic agent for type-2 diabetes. The effects of the individual mutations, however, have not yet been demonstrated due to the lack of experimentally determined structural information. Here we describe three crystal structures of the first isolated R-ATA, its G136F mutant and engineered ATA-117-Rd11, which indicated that the mutation introduced into the 136th residue altered the conformation of a loop next to the active site, resulting in a substrate-binding site with drastically modified volume, shape, and surface properties, to accommodate the large pro-sitagliptin ketone. Our findings provide a detailed explanation of the previously reported molecular engineering of ATA-117-Rd11 and propose that the loop near the active site is a new target for the rational design to change the substrate specificity of ATAs.

Molecular mechanism of distinct salt-dependent enzyme activity of two halophilic nucleoside diphosphate kinases.

Nucleoside diphosphate kinases from haloarchaea Haloarcula quadrata (NDK-q) and H. sinaiiensis (NDK-s) are identical except for one out of 154 residues, i.e., Arg(31) in NDK-q and Cys(31) in NDK-s. However, the salt-dependent activity profiles of NDK-q and NDK-s are quite different: the optimal NaCl concentrations of NDK-q and NDK-s are 1 M and 2 M, respectively. We analyzed the relationships of the secondary, tertiary, and quaternary structures and NDK activity of these NDKs at various salt concentrations, and revealed that 1), NDK-q is present as a hexamer under a wide range of salt concentrations (0.2-4 M NaCl), whereas NDK-s is present as a hexamer at an NaCl concentration above 2 M and as a dimer at NaCl concentrations below 1 M; 2), dimeric NDK-s has lower activity than hexameric NDK-s; and 3), dimeric NDK-s has higher helicity than hexameric NDK-s. We also determined the crystal structure of hexameric NDK-q, and revealed that Arg(31) plays an important role in stabilizing the hexamer. Thus the substitution of Arg (as in NDK-q) to Cys (as in NDK-s) at position 31 destabilizes the hexameric assembly, and causes dissociation to less active dimers at low salt concentrations.

A unique catalytic triad revealed by the crystal structure of APE0912, a short-chain dehydrogenase/reductase family protein from Aeropyrum pernix K1.

Short-chain dehydrogenases/reductases (SDRs), about 250-residue long, are enzymes that catalyze NAD(P)(H)-dependent oxidation/reduction of various substrates such as alcohols, sugars, steroids, aromatic compounds, and xenobiotics.1–3 SDR family proteins have the N-terminal Rossman fold made up of 7 to 8 b-strands, which binds the cofactor NAD(P)(H), and a catalytic triad composed of Ser-Tyr-Lys.4 The APE0912 gene of hyperthermophilic archaea Aeropyrum pernix K1 encodes an SDR family protein with an unknown function.5 We have determined the crystal structure of APE0912 at 1.8-Å resolution. The overall structure of APE0912 is similar to those of other SDR family members, but APE0912 does not have a catalytic triad Ser-Tyr-Lys characteristic of the SDR family members. Instead, APE0912 has a unique Ser-Ser-Arg triad. The two molecules in the asymmetric unit exhibit open and closed forms that alter the accessibility of substrates and cofactors to the Ser-SerArg triad. Here, we report the crystal structure of APE0912 with its unique catalytic triad and the structural difference between open and closed forms.

Isolation and characterization of benzo[a]pyrene-degrading bacteria from the Tokyo Bay area and Tama River in Japan.

Benzo[a]pyrene (BaP) is one of the polycyclic aromatic hydrocarbons, and has serious detrimental effects on human health and aquatic environments. In this study, we isolated nine bacterial strains capable of degrading BaP from the Tokyo Bay area and Tama River in Japan. The isolated bacteria belonged to the phyla Actinobacteria, Firmicutes, Proteobacteria and Bacteroidetes, indicating that the BaP-degrading bacteria were widely present in the hydrosphere. ITB11, which shared 100% 16S rRNA identity with Mesoflavibacter zeaxanthinifaciens in the phylum Bacteroidetes, showed the highest degradation of BaP (approximately 86%) among the nine isolated strains after 42 days. Moreover, it was found that three of the nine isolated strains collectively removed 50-55% of BaP during the first 7 days. Growth measurement of M. zeaxanthinifaciens revealed that the strain utilized BaP as a sole carbon and energy source and salicylate acted only as an inducer of BaP degradation.

Crystal structure of dibenzothiophene sulfone monooxygenase BdsA from Bacillus subtilis WU-S2B

The dibenzothiophene (DBT) sulfone monooxygenase BdsA from Bacillus subtilis WU‐S2B catalyzes the conversion of DBT sulfone to 2′‐hydroxybiphenyl 2‐sulfinate. We report the crystal structures of BdsA at a resolution of 2.80 Å. BdsA exists as a homotetramer with a dimer‐of‐dimers configuration in the crystal, and the interaction between E288 and R296 in BdsA is important for tetramer formation. A structural comparison with homologous proteins shows that the orientation and location of the α9‐α12 helices in BdsA are closer to those of the closed form than those of the open form in the EDTA monooxygenase EmoA. Proteins 2017; 85:1171–1177.

Crystallization and preliminary X-ray analysis of γ-­hexachlorocyclohexane dehydrochlorinase LinA from Sphingobium japonicum UT26

LinA from Sphingobium japonicum UT26 catalyzes two steps of dehydrochlorination from gamma-hexachlorocyclohexane (gamma-HCH) to 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN) via gamma-pentachlorocyclohexene (gamma-PCCH). LinA was crystallized by the sitting-drop vapour-diffusion method using PEG 3350 as the precipitant. The crystals belonged to space group P4(1) or P4(3), with unit-cell parameters a = b = 68.9, c = 101.9 A, and diffracted X-rays to 2.25 A resolution. The crystal contained three molecules in the asymmetric unit.