Crystal structure of Streptomyces olivaceoviridis E-86 beta-xylanase containing xylan-binding domain

Xylanases hydrolyse the beta-1,4-glycosidic bonds within the xylan backbone and belong to either family 10 or 11 of the glycoside hydrolases, on the basis of the amino acid sequence similarities of their catalytic domains. Generally, xylanases have a core catalytic domain, an N and/or C-terminal substrate-binding domain and a linker region. Until now, X-ray structural analyses of family 10 xylanases have been reported only for their catalytic domains and do not contain substrate-binding domains. We have determined the crystal structure of a family 10 xylanase containing the xylan-binding domain (XBD) from Streptomyces olivaceoviridis E-86 at 1.9 A resolution. The catalytic domain comprises a (beta/alpha)(8)-barrel topologically identical to other family 10 xylanases. XBD has three similar subdomains, as suggested from a triple-repeat sequence, which are assembled against one another around a pseudo-3-fold axis, forming a galactose-binding lectin fold similar to ricin B-chain. The Gly/Pro-rich linker region connecting the catalytic domain and XBD is not visible in the electron density map, probably because of its flexibility. The interface of the two domains in the crystal is hydrophilic, where five direct hydrogen bonds and water-mediated hydrogen bonds exist. The sugar-binding residues seen in ricin/lactose complex are spatially conserved among the three subdomains in XBD, suggesting that all of the subdomains in XBD have the capacity to bind sugars. The flexible linker region enables the two domains to move independently and may provide a triple chance of substrate capturing and catalysis. The structure reported here represents an example where the metabolic enzyme uses a ricin-type lectin motif for capturing the insoluble substrate and promoting catalysis.     

Structure and function of a family 10 beta-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.     

Structure-based engineering of glucose specificity in a family 10 xylanase from Streptomyces olivaceoviridis E-86

Substrate specificity is one of the most important functional property of enzymes. We use family 10 xylanase from Streptomyces olivaceoviridis as a model for substrate specificity of glycoside hydrolases. Seven variants were initially designed to change the preference from xylose to glucose at substrate binding subsites ?2 and ?1. The known mobility of Trp at the ?1 subsite and the influence of its environment, which is different in subset 1 and subset 2 family 10 enzymes, were taken into account in variant design. Q88A/R275A had the best ratio of p-nitrophenyl cellobioside vs p-nitrophenyl xylobioside hydrolyzing activity in the first series of variants. The crystal structure shows a movement of Trp274 compared to the native, as a result of loss of interaction with the long side chain of Arg275. The movement creates extra space for the hydroxymethyl of glucose, resulting in improved K_m on glucose derived substrates, while the negative effect on k_cat is compensated by the Q88A mutation, which also contributes to a further reduction of K_m. Further mutagenesis based on the Q88A/R275A variant resulted in 5.2 times improvement compared to the wild-type p-nitrophenyl cellobioside hydrolyzing activity, which is the best improvement obtained so far for an engineered xylanase.     

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 alpha-l-arabinofuranosyl- and 4-O-methyl-alpha-d-glucuronosyl-xylooligosaccharides. The bound sugars were observed in the subsites of the catalytic cleft and also in subdomains alpha and gamma 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, 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.     

High activity xylanase production by Streptomyces olivaceoviridis E-86

The effects of different factors on xylanase production by Streptomyces olivaceoviridis E-86 were studied under shake flask conditions. The best initial pH value of growth medium for xylanase production was pH 6.0. Corn cob xylan and beef peptone were the best C source and N source, respectively. The enzyme activity was doubled by addition of 1.5% (v/v) Tween-80 in the medium. By the combination of the above variables, the highest xylanase activity obtained was 1653 U/ml which is the highest ever reported from Streptomyces sp.     

Novel sugar-binding specificity of the type XIII xylan-binding domain of a family F/10 xylanase from Streptomyces olivaceoviridis E-86

The type XIII xylan-binding domain (XBD) of a family F/10 xylanase (FXYN) from Streptomyces olivaceoviridis E-86 was found to be structurally similar to the ricin B chain which recognizes the non-reducing end of galactose and specifically binds to galactose containing sugars. The crystal structure of XBD [Fujimoto, Z. et al. (2000) J. Mol. Biol. 300, 575-585] indicated that the whole structure of XBD is very similar to the ricin B chain and the amino acids which form the galactose-binding sites are highly conserved between the XBD and the ricin B chain. However, our investigation of the binding abilities of wt FXYN and its truncated mutants towards xylan demonstrated that the XBD bound xylose-based polysaccharides. Moreover, it was found that the sugar-binding unit of the XBD was a trimer, which was demonstrated in a releasing assay using sugar ranging in size from xylose to xyloheptaose. These results indicated that the binding specificity of the XBD was different from those of the same family lectins such as the ricin B chain. Somewhat surprisingly, it was found that lactose could release the XBD from insoluble xylan to a level half of that observed for xylobiose, indicating that the XBD also possessed the same galactose recognition site as the ricin B chain. It appears that the sugar-binding pocket of the XBD has evolved from the ancient ricin super family lectins to bind additional sugar targets, resulting in the differences observed in the sugar-binding specificities between the lectin group (containing the ricin B chain) and the enzyme group.     

Subunit composition of a large xylanolytic complex (xylanosome) from Streptomyces olivaceoviridis E-86

A xylanolytic complex (xylanosome) was isolated from Streptomyces olivaceoviridis E-86 grown on corncob xylan. The isolated xylanosome exhibited a high molecular mass of approximately 3.8 x 10(7) Da (weight average) using size exclusion chromatography/multi-angle laser light scattering (SEC/MALLS), and was composed of at least 8 subunits with a mass range from 12 to 60 kDa. When a SDS-polyacrylamide gel zymogram was examined, the subunits of 47, 35, 32, and 23 kDa were found to have xylanase activity, while the 30-kDa subunit had CMCase activity. According to N-terminal sequence analyses, the 47- and 23-kDa subunits were found to be identical to the two reported xylanases, namely FXYN and GXYN, of S. olivaceoviridis E-86. Both the 35- and 32-kDa subunits were found to be truncated forms of the intact FXYN xylanase that possibly resulted from the degradation by proteases. The 15-kDa subunit consisted solely the xylan-binding domain of the FXYN xylanase. The purified xylanosome appeared to bind partially to xylan and poorly to Avicel.     

Module shuffling of a family F/10 xylanase: replacement of modules M4 and M5 of the FXYN of Streptomyces olivaceoviridis E-86 with those of the Cex of Cellulomonas fimi

To facilitate an understanding of structure-function relationships, chimeric xylanases were constructed by module shuffling between the catalytic domains of the FXYN from Streptomyces olivaceoviridis E-86 and the Cex from Cellulomonas fimi. In the family F/10 xylanases, the modules M4 and M5 relate to substrate binding so that modules M4 and M5 of the FXYN were replaced with those of the Cex and the chimeric enzymes denoted FCF-C4, FCF-C5 and FCF-C4,5 were constructed. The k(cat) value of FCF-C5 for p-nitrophenyl-beta-D-cellobioside was similar to that of the FXYN (2.2 s(-1)); however, the k(cat) value of FCF-C4 for p-nitrophenyl-beta-D-cellobioside was significantly higher (7.0 s(-1)). The loss of the hydrogen bond between E46 and S22 or the presence of the I49W mutation would be expected to change the position of Q88, which plays a pivotal role in discriminating between glucose and xylose, resulting in the increased k(cat) value observed for FCF-C4 acting on p-nitrophenyl-beta-D-cellobioside since module M4 directly interacts with Q88. To investigate the synergistic effects of the different modules, module M10 of the FCF-C4 chimera was replaced with that of the Cex. The effects of replacement of module M4 and M10 were almost additive with regard to the K:(m) and k(cat) values.     

An investigation of the nature and function of module 10 in a family F/10 xylanase FXYN of Streptomyces olivaceoviridis E-86 by module shuffling with the Cex of Cellulomonas fimi and by site-directed mutagenesis

Although the amino acid homology in the catalytic domain of FXYN xylanase from Streptomyces olivaceoviridis E-86 and Cex xylanase from Cellulomonas fimi is only 50%, an active chimeric enzyme was obtained by replacing module 10 in FXYN with module 10 from Cex. In the family F/10 xylanases, module 10 is an important region as it includes an acid/base catalyst and a substrate binding residue. In FXYN, module 10 consists of 15 amino acid residues, while in Cex it consists of 14 amino acid residues. The Km and kcat values of the chimeric xylanase FCF-C10 for PNP-xylobioside (PNP-X2) were 10-fold less than those for FXYN. CD spectral data indicated that the structure of the chimeric enzyme was similar to that of FXYN. Based on the comparison of the amino acid sequences of FXYN and Cex in module 10, we constructed four mutants of FXYN. When D133 or S135 of FXYN was deleted, the kinetic properties were not changed from those of FXYN. By deletion of both D133 and S135, the Km value for PNP-X2 decreased from the 2.0 mM of FXYN to 0.6 mM and the kcat value decreased from the 20 s(-1) of FXYN to 8.7 s(-1). Insertion of Q140 into the doubly deleted mutant further reduced the Km value to 0.3 mM and the kcat value to 3.8 s(-1). These values are close to those for the chimeric enzyme FCF-C10. These results indicate that module 10 itself is able to accommodate changes in the sequence position of amino acids which are critical for enzyme function. Since changes of the spatial position of these amino acids would be expected to result in enzyme inactivation, module 10 must have some flexibility in its tertiary structure. The structure of module 10 itself also affects the substrate specificity of the enzyme.     

橄榄绿链霉菌细胞膜中几丁单、寡糖结合蛋白质的分离、纯化和结合动力学

橄榄绿链霉菌细胞膜中存在能与N 乙酰葡糖胺特异性结合的蛋白质。采用超声波破碎、超速离心等方法 ,分离得到细胞膜后用TritonX 10 0抽提 ,经连续的阴离子层析后 ,可分别得到能与N 乙酰葡糖胺特异性结合的 46 .0kD和 47.5kD蛋白质。底物竞争结合实验证实 ,这两种蛋白质还能与其他几丁质降解产物 (几丁二糖至几丁六糖 )特异性结合而不与葡萄糖、纤维二糖等结合。采用介质表面感应技术 (surfaceplasmonresonance)检测出46kD蛋白质与几丁单糖至几丁六糖的解离常数 (Kd)在 8.2 9× 10 -9～ 1.95× 10 -5mol/L之间 ,单糖最高而三糖最低。决定其差异的主要因子是底物结合速率 (Kon)而非解离速率 (Koff)。N末端氨基酸序列分析结果推测 ,该蛋白质属于一个潜在的ATP结合转运系统     

Purification and characterization of a novel aminoacylase from Streptomyces mobaraensis

A novel aminoacylase was purified to homogeneity from culture broth of Streptomyces mobaraensis, as evidenced by SDS-polyacrylamide gel electrophoresis (PAGE). The enzyme was a monomer with an approximate molecular mass of 100 kDa. The purified enzyme was inhibited by the presence of 1,10-phenanthroline and activated by the addition of Co2+. It was stable at temperatures of up to 60 degrees C for 1 h at pH 7.2. It showed broad substrate specificity to N-acetylated L-amino acids. It catalyzed the hydrolysis of the amide bonds of various N-acetylated L-amino acids, except for Nepsilon-acetyl-L-lysine and N-acetyl-L-proline. Hydrolysis of N-acetyl-L-methionine and N-acetyl-L-histidine followed Michaelis-Menten kinetics with K(m) values of 1.3+/-0.1 mM and 2.7+/-0.1 mM respectively. The enzyme also catalyzed the deacetylation of 7-aminocephalosporanic acid (7-ACA) and cephalosporin C. Moreover, feruloylamino acids and L-lysine derivatives of ferulic acid derivatives were synthesized in an aqueous buffer using the enzyme.     

Purification and characterization of a chitosanase from Streptomyces N174

A highly efficient chitosanase producer, the actinomycete N174, identified by chemotaxonomic methods as belonging to the genus Streptomyces was isolated from soil. Chitosanase production by N174 was inducible by chitosan or d-glucosamine. In culture filtrates the chitosanase accounted for 50–60% of total extracellular proteins. The chitosanase was purified by polyacrylic acid precipitation, CM-Sepharose and gel permeation chromatography. The maximum velocity of chitosan degradation was obtained at 65° C when the pH was maintained at 5.5. The enzyme degraded chitosans with a range of acetylation degrees from 1 to 60% but not chitin or CM-cellulose. The enzyme showed an endo-splitting type of activity and the end-product of chitosan degradation contained a mixture of dimers and trimers of d-glucosamine.Correspondence to: R. Brzezinski     

Purification and characterization of a hygromycin B phosphotransferase from Streptomyces hygroscopicus

A hygromycin B phosphotransferase activity from Streptomyces hygroscopicus has been highly purified by ammonium sulphate fractionation followed by affinity column chromatography through Sepharose-6B-hygromycin-B. The combined active fractions showed a single protein band (41 kDa) when subjected to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. When gel electrophoresis was performed under non-denaturing conditions, the single protein band promoted in situ phosphorylation of hygromycin B, indicating that this protein corresponded to the purified hygromycin B phosphotransferase. The enzyme has been purified 236-fold and approximate Km values of 0.56 microM for hygromycin B and ATP, respectively, were deduced.     

Purification and partial characterization of a cholesterol oxidase from Streptomyces fradiae

An extracellular cholesterol oxidase from Streptomyces fradiae (PTCC 1121) was purified in one step using DEAE-Sepharose. The purified enzyme had a molecular weight of 60 KDa. The optimum pH and temperature for activity was found to be 7 and 70 degrees C, respectively. This cholesterol oxidase was stable in pHs between 4-10 at 4 degrees C until 4 h. Thermal stability experiments showed that it has high stability and retains its full activity at 50 degrees C for 90 min. K(m) value for cholesterol oxidase was obtained to be about 7.06 x 10(-)(5) Mol.     

Purification and characterization of a 7Fe ferredoxin from Streptomyces griseus

A ferredoxin has been purified from Streptomyces griseus grown in soybean flour-containing medium. The homogeneous protein has a molecular weight near 14000 as determined by both PAGE and size exclusion chromatography. The iron and labile sulfide content is 6–7 atoms/mole protein. EPR spectroscopy of native S. griseus ferredoxin shows an isotropic signal at g=2.01 which is typical of [3Fe-4S]¹⁺ clusters and which quantitates to 0.9 spin/mole. Reduction of the ferredoxin by excess dithionite at pH 8.0 produces an EPR silent state with a small amount of a g=1.95 type signal. Photoreduction in the presence of deazaflavin generates a signal typical of [4Fe-4S]¹⁺ clusters at much higher yields (0.4–0.5 spin/mole) with major features at g-values of 2.06, 1.94, 1.90 and 1.88. This latter EPR signal is most similar to that seen for reduced 7Fe ferredoxins, which contain both a [3Fe-4S] and [4Fe-4S] cluster. In vitro reconstitution experiments demonstrate the ability of the S. grisues ferredoxin to couple electron transfer between spinach ferredoxin reductase and S. griseus cytochrome P-450_soy for NADPH-dependent substrate oxidation. This represents a possible physiological function for the S. griseus ferredoxin, which if true, would be the first functional role demonstrated for a 7Fe ferredoxin.     

Purification and partial characterization of polygalacturonase from Streptomyces lydicus

Polygalacturonase produced by Streptomyces lydicus was purified to homogeneity by ultrafiltration and a combination of ion exchange and gel filtration chromatographic procedures. The purified enzyme was an exo-polygalacturonase with a molecular weight of 43 kDa. It was optimally active at 50 degrees C and pH 6.0. The enzyme was stable from pH 4.0 to 7.0 and at or below 45 degrees C for 90 min. K(m) value for polygalacturonic acid was 1.63 mg/mL and the corresponding V(max) was 677.8 microM min(-1) mg(-1). The inhibition constant (K(i)) for gluconic acid d-lactone was 20.75 mM. Purified enzyme had been inhibited by N-bromosuccinimide, while l-tryptophan could induce enzyme activity, indicating the involvement of tryptophan at the active site.     

Purification and characterization of tryptophan dioxygenase from Streptomyces parvulus

Tryptophan dioxygenase, derived from Streptomyces parvulus, was purified to near homogeneity and shown to have a native Mr of 88,000. Kinetic parameters of the enzyme were determined and evidence suggesting that it is a hemoprotein was obtained. Tryptophan dioxygenase has a high specificity toward L-tryptophan with an apparent Km of 0.3 mM. L-3-Hydroxykynurenine was a competitive inhibitor with respect to L-tryptophan with a Ki of 0.16 mM. In vitro, the enzyme displayed little activity in the absence of a reducing agent; ascorbate, at 50 mM, was the preferred reductant providing almost a 50-fold increase in enzyme activity. The regulation of tryptophan dioxygenase synthesis and activity is described. The expression of the enzyme is correlated with the biosynthesis of actinomycin D in S. parvulus. These results support the hypothesis that tryptophan dioxygenase functions as the first enzyme in the sequence converting L-tryptophan to the chromophore of this antibiotic.