Hiroshi Mizuno

Crystal structures of decorated xylooligosaccharides bound to a family 10 xylanase from Streptomyces olivaceoviridis E-86

The family 10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) consists of a GH10 catalytic domain, which is joined by a Gly/Pro-rich linker to a family 13 carbohydrate-binding module (CBM13) that interacts with xylan. To understand how GH10 xylanases and CBM13 recognize decorated xylans, the crystal structure of SoXyn10A was determined in complex with α-l-arabinofuranosyl- and 4-O-methyl-α-d-glucuronosyl-xylooligosaccharides. The bound sugars were observed in the subsites of the catalytic cleft and also in subdomains α and γ of CBM13. The data reveal that the binding mode of the oligosaccharides in the active site of the catalytic domain is entirely consistent with the substrate specificity and, in conjunction with the accompanying paper (Pell, G., Taylor, E. J., Gloster, T. M., Turkenburg, J. P., Fontes, C. M. G. A., Ferreira, L. M. A., Nagy, T., Clark, S. J., Davies, G. J., and Gilbert, H. J. (2004) J. Biol. Chem. 279, 9597-9605), demonstrate that the accommodation of the side chains in decorated xylans is conserved in GH10 xylanases of SoXyn10A against arabinoglucuronoxylan. CBM13 was shown to bind xylose or xylooligosaccharides reversibly by using nonsymmetric sugars as the ligands. The independent multiple sites in CBM13 may increase the probability of substrate binding.

Structure and Function of a Family 10 β-Xylanase Chimera of Streptomyces olivaceoviridis E-86 FXYN and Cellulomonas fimi Cex

The catalytic domain of xylanases belonging to glycoside hydrolase family 10 (GH10) can be divided into 22 modules (M1 to M22; Sato, Y., Niimura, Y., Yura, K., and Go, M. (1999) Gene (Amst.) 238, 93-101). Inspection of the crystal structure of a GH10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) revealed that the catalytic domain of GH10 xylanases can be dissected into two parts, an N-terminal larger region and C-terminal smaller region, by the substrate binding cleft, corresponding to the module border between M14 and M15. It has been suggested that the topology of the substrate binding clefts of GH10 xylanases are not conserved (Charnock, S. J., Spurway, T. D., Xie, H., Beylot, M. H., Virden, R., Warren, R. A. J., Hazlewood, G. P., and Gilbert, H. J. (1998) J. Biol. Chem. 273, 32187-32199). To facilitate a greater understanding of the structure-function relationship of the substrate binding cleft of GH10 xylanases, a chimeric xylanase between SoXyn10A and Xyn10A from Cellulomonas fimi (CfXyn10A) was constructed, and the topology of the hybrid substrate binding cleft established. At the three-dimensional level, SoXyn10A and CfXyn10A appear to possess 5 subsites, with the amino acid residues comprising subsites -3 to +1 being well conserved, although the +2 subsites are quite different. Biochemical analyses of the chimeric enzyme along with SoXyn10A and CfXyn10A indicated that differences in the structure of subsite +2 influence bond cleavage frequencies and the catalytic efficiency of xylooligosaccharide hydrolysis. The hybrid enzyme constructed in this study displays fascinating biochemistry, with an interesting combination of properties from the parent enzymes, resulting in a low production of xylose.

Oxydemeton-Methyl Resistance, Mechanisms, and Associated Fitness Cost in Green Peach Aphids (Hemiptera: Aphididae)

Abstract Susceptibility to oxydemeton-methyl and imidacloprid, and the inhibitory effects of oxydemeton-methyl and some organophosphate compounds on acetylcholinesterase (AChE) and carboxylesterase activity were studied in two populations (Karaj and Rasht) of green peach aphids, Myzus persicae (Sulzer). Results show that the Karaj population was resistant to oxydemeton-methyl but susceptible to imidacloprid. The esterase activity of the resistant and susceptible populations suggests that one of the resistance mechanisms to oxydemeton-methyl was esterase-based. The inhibition assay shows that the AChE of the Karaj population is less sensitive to oxydemeton-methyl and paraoxon derivatives. Regarding the paraoxon derivatives, the smaller paraoxon side chain is more potent against the modified AChE than against the AChE from the susceptible strain. Fertility life table parameters of green peach aphid populations resistant and susceptible to oxydemeton-methyl also were studied under laboratory conditions. The standard errors of the population growth parameters were calculated using the Jackknife method. Results showed that susceptible strain exhibits a significantly higher rm than the resistant strain, probably because the resistant strain had a higher generation time than the susceptible strain. These results suggested that the resistant Karaj strain may be less fit than the susceptible strain.

Purification, crystallization and preliminary X-ray crystallographic study of α-amylase from Bacillus stearothermophilus

A recombinant alpha-amylase from Bacillus stearothermophilus was found to be produced as several isoforms arising from different N--terminal processing. Some of those isoforms were purified to homogeneity and crystallized at 293 K using the hanging-drop vapour-diffusion method under the following conditions: 35 mM sodium acetate (pH 4.6), 6.25%(v/v) 2-propanol, in the presence of 1.23%(w/v) acarbose (a pseudo-oligosaccharide inhibitor) in the drop. The crystals diffracted beyond 2.0 A resolution using synchrotron radiation at the Photon Factory, Tsukuba. They belong to the monoclinic space group P2(1), with unit-cell parameters a = 53.7 (2), b = 92.9 (4), c = 53.2 (2) A, beta = 109.4 (1) degrees.

Crystallization and preliminary X-ray studies of flavocetin-A, a platelet glycoprotein Ib-binding protein from the habu snake venom

Flavocetin-A (FL-A) is a platelet glycoprotein Ib-binding protein, a high molecular mass oligomer (149 kDa) of C-type lectin-like subunits alpha and beta isolated from the habu snake venom. Purified FL-A crystallized in the tetragonal space group I4 with unit-cell dimensions a = b = 121.0, c = 63.2 A. The crystals diffract to at least 2.4 A resolution. The structure has been solved by molecular replacement using the crystal structure of factors IX/X-binding protein (PDB code 1ixx) as a search model. The asymmetric unit contains one heterodimer, showing that FL-A is a novel tetradimer (alphabeta)(4) composed of four heterodimers related by a crystallographic fourfold axis.

Purification, crystallization and preliminary X-ray crystallographic analysis of human CCG1-interacting factor B.

A novel human factor CIB (CCG1-interacting factor B) has been isolated using the yeast two-hybrid system. The 22 kDa CIB protein has been expressed in Escherichia coli, purified to homogeneity and crystallized in a form suitable for crystallographic studies. The protein was crystallized in the orthogonal space group P2(1)2(1)2(1), with unit-cell parameters a = 43.60 (2), b = 44.45 (1), c = 110.70 (5) A and one molecule in the asymmetric unit. The crystal diffracted beyond 2.2 A resolution using synchrotron radiation.