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1.
dTDP-6-deoxy-d-allose, an unusual deoxysugar, has been identified as an intermediate in the mycinose biosynthetic pathway of several macrolide antibiotics. In order to characterize the biosynthesis of this deoxysugar, we have cloned and heterologously overexpressed gerK1 in Escherichia coli BL21 (DE3) cells. This gene encodes for a protein with the putative function of a dTDP-4-keto-6-deoxyglucose reductase, which appears to be involved in the dihydrochalcomycin (GERI-155) biosynthesis evidenced by Streptomyces sp KCTC 0041BP. Our results revealed that GerK1 exhibited a specific reductive effect on the 4-keto carbon of dTDP-4-keto-6-deoxy-d-allose, with the hydroxyl group in an axial configuration at the C3 position only. The enzyme catalyzed the conversion of dTDP-4-keto-6-deoxyglucose to dTDP-6-deoxy-beta-D-allose, according to the results of an in vitro coupled enzyme assay, in the presence of GerF (dTDP-4-keto-6-deoxyglucose 3-epimerase). The product was isolated, and its stereochemistry was determined via nuclear magnetic resonance analysis. 相似文献
2.
Minna M?ki Nina J?rvinen Jarkko R?bin? Christophe Roos Hannu Maaheimo Risto Renkonen 《European journal of biochemistry》2002,269(2):593-601
Pseudomonas aeruginosa is an opportunistic Gram-negative bacterium that causes severe infections in a number of hosts from plants to mammals. A-band lipopolysaccharide of P. aeruginosa contains d-rhamnosylated O-antigen. The synthesis of GDP-D-rhamnose, the d-rhamnose donor in d-rhamnosylation, starts from GDP-D-mannose. It is first converted by the GDP-mannose-4,6-dehydratase (GMD) into GDP-4-keto-6-deoxy-D-mannose, and then reduced to GDP-D-rhamnose by GDP-4-keto-6-deoxy-D-mannose reductase (RMD). Here, we describe the enzymatic characterization of P. aeruginosa RMD expressed in Saccharomyces cerevisiae. Previous success in functional expression of bacterial gmd genes in S. cerevisiae allowed us to convert GDP-D-mannose into GDP-4-keto-6-deoxy-D-mannose. Thus, coexpression of the Helicobacter pylori gmd and P. aeruginosa rmd genes resulted in conversion of the 4-keto-6-deoxy intermediate into GDP-deoxyhexose. This synthesized GDP-deoxyhexose was confirmed to be GDP-rhamnose by HPLC, matrix-assisted laser desorption/ionization time-of-flight MS, and finally NMR spectroscopy. The functional expression of P. aeruginosa RMD in S. cerevisiae will provide a tool for generating GDP-rhamnose for in vitro rhamnosylation of glycoprotein and glycopeptides. 相似文献
3.
Nao Suzuki Yoshio Nakano Yasuo Yoshida Takashi Nezu Yoshihiro Terada Yoshihisa Yamashita Toshihiko Koga 《European journal of biochemistry》2002,269(23):5963-5971
The serotype a-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans is an unusual sugar, 6-deoxy-d-talose. Guanosine diphosphate (GDP)-6-deoxy-d-talose is the activated sugar nucleotide form of 6-deoxy-d-talose, which has been identified as a constituent of only a few microbial polysaccharides. In this paper, we identify two genes encoding GDP-6-deoxy-d-talose synthetic enzymes, GDP-alpha-d-mannose 4,6-dehydratase and GDP-4-keto-6-deoxy-d-mannose reductase, in the gene cluster required for the biosynthesis of serotype a-specific polysaccharide antigen from A. actinomycetemcomitans SUNYaB 75. Both gene products were produced and purified from Escherichia coli transformed with plasmids containing these genes. Their enzymatic reactants were analysed by reversed-phase HPLC (RP-HPLC). The sugar nucleotide produced from GDP-alpha-d-mannose by these enzymes was purified by RP-HPLC and identified by electrospray ionization-MS, 1H nuclear magnetic resonance, and GC/MS. The results indicated that GDP-6-deoxy-d-talose is produced from GDP-alpha-d-mannose. This paper is the first report on the GDP-6-deoxy-d-talose biosynthetic pathway and the role of GDP-4-keto-6-deoxy-d-mannose reductase in the synthesis of GDP-6-deoxy-d-talose. 相似文献
4.
Alderwick LJ Dover LG Seidel M Gande R Sahm H Eggeling L Besra GS 《Glycobiology》2006,16(11):1073-1081
The arabinogalactan (AG) of Corynebacterianeae is a critical macromolecule that tethers mycolic acids to peptidoglycan, thus forming a highly impermeable cell wall matrix termed the mycolyl-arabinogalactan peptidoglycan complex (mAGP). The front line anti-tuberculosis drug, ethambutol (Emb), targets the Mycobacterium tuberculosis and Corynebacterium glutamicum arabinofuranosyltransferase Mt-EmbA, Mt-EmbB and Cg-Emb enzymes, respectively, which are responsible for the biosynthesis of the arabinan domain of AG. The substrate utilized by these important glycosyltransferases, decaprenylmonophosphoryl-D-arabinose (DPA), is synthesized via a decaprenylphosphoryl-5-phosphoribose (DPPR) synthase (UbiA), which catalyzes the transfer of 5-phospho-ribofuranose-pyrophosphate (pRpp) to decaprenol phosphate to form DPPR. Glycosyl compositional analysis of cell walls extracted from a C. glutamicum::ubiA mutant revealed a galactan core consisting of alternating beta(1-->5)-Galf and beta(1-->6)-Galf residues, completely devoid of arabinan and a concomitant loss of cell-wall-bound mycolic acids. In addition, in vitro assays demonstrated a complete loss of arabinofuranosyltransferase activity and DPA biosynthesis in the C. glutamicum::ubiA mutant when supplemented with p[14C]Rpp, the precursor of DPA. Interestingly, in vitro arabinofuranosyltransferase activity was restored in the C. glutamicum::ubiA mutant when supplemented with exogenous DP[14C]A substrate, and C. glutamicum strains deficient in ubiA, emb, and aftA all exhibited different levels of DPA biosynthesis. 相似文献
5.
Glycine is an amino acid tasting sweet to humans. In 2-bottle tests, C57BL/6ByJ (B6) mice strongly prefer glycine solutions, whereas 129P3/J (129) mice do not, suggesting that they differ in perception of glycine taste. We examined this question using the conditioned taste aversion (CTA) generalization technique. CTA was achieved by injecting LiCl after drinking glycine, and next its generalization to 10 taste solutions (glycine, sucrose, saccharin, D-tryptophan, L-tryptophan, L-alanine, L-proline, L-glutamine, NaCl, and HCl) was examined by video recording licking behavior. Both B6 and 129 mice generalized the aversion to sucrose, saccharin, L-alanine, and L-proline and did not generalize it to NaCl, HCl, and L-tryptophan. This indicates that both B6 and 129 mice perceive the sweetness (i.e., a sucrose-like taste) of glycine. Thus, the lack of a glycine preference by 129 mice cannot be explained by their inability to perceive its sweetness. Strain differences were observed for CTA generalization to 2 amino acids: 129 mice generalized aversion to L-glutamine but not D-tryptophan, whereas B6 mice generalized it to D-tryptophan but not L-glutamine. 129.B6-Tas1r3 congenic mice with 2 genotypes of the Tas1r3 locus (B6/129 heterozygotes and 129/129 homozygotes) did not differ in aversion generalization, suggesting that the differences between 129 and B6 strains are not attributed to the Tas1r3 allelic variants and that other, yet unknown, genes are involved in taste perception of amino acids. 相似文献
6.
Suzuki N Nakano Y Yoshida Y Nakao H Yamashita Y Koga T 《Biochimica et biophysica acta》2000,1517(1):135-138
The serotype a-specific polysaccharide antigen (SPA) of Actinobacillus actinomycetemcomitans consists of 6-deoxy-D-talose. A gene cluster associated with the biosynthesis of SPA was cloned and sequenced from the chromosomal DNA of A. actinomycetemcomitans SUNYaB 75 (serotype a). This cluster consisted of 14 open reading frames. Insertional inactivation of eight genes in this cluster resulted in loss of the ability of A. actinomycetemcomitans SUNYaB 75 cells to produce the polysaccharide. A protein database search revealed that the 11 sequential genes containing these eight genes were not found in SPA-associated gene clusters of the other serotypes of A. actinomycetemcomitans. These results suggest that the gene cluster is unique to serotype a and is essential to the synthesis of the SPA. 相似文献
7.
The 6-deoxyhexose L-fucose is an important and characteristic element in glycoconjugates of bacteria (e.g., lipopolysaccharides), plants (e.g., xyloglucans) and animals (e.g., glycolipids, glycoproteins, and oligosaccharides). The biosynthetic pathway of GDP-L-fucose starts with a dehydration of GDP-D-mannose catalyzed by GDP-D-mannose 4,6-dehydratase (Gmd) creating GDP-4-keto-6-deoxymannose which is subsequently converted by the GDP-4-keto-6-deoxy-D-mannose 3,5-epimerase-4-reductase (WcaG; GDP-beta-L-fucose synthetase) to GDP-beta-L-fucose. Both biosynthetic genes gmd and wcaG were cloned from Escherichia coli K12 and the enzymes overexpressed under control of the T7 promoter in the expression vectors pET11a and pET16b, yielding both native and N-terminal His-tag fusion proteins, respectively. The activities of the Gmd and WcaG were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-beta-L-fucose was optimized and the final product was purified. The formation of GDP-beta-L-fucose by the recombinant enzymes was verified by HPLC and NMR analyses. The His-tag fusion variants of the Gmd and WcaG proteins were purified to near homogeneity. The His-tag Gmd recombinant enzyme was inactive, whereas His-tag WcaG showed very similar enzymatic properties relative to the native GDP-beta-L-fucose synthetase. With the purified His-tag WcaG Km and Vmax values, respectively, of 40 microM and 23 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose and of 21 microM and 10 nkat/mg protein for the cosubstrate NADPH were obtained; a pH optimum of 7.5 was determined and the enzyme was stimulated to equal extend by the divalent cations Mg2+ and Ca2+. The Gmd enzyme showed a strong feedback inhibition by GDP-beta-L-fucose. 相似文献
8.
GDP-N-acetyl-d-perosamine is a precursor of the LPS-O-antigen biosynthesis in Escherichia coli O157:H7. Like other GDP-6-deoxyhexoses, GDP-N-acetyl-d-perosamine is supposed to be synthesized via GDP-4-keto-6-deoxy-d-mannose, followed by a transamination- and an acetylation-reaction catalyzed by PerA and PerB. In this study, we have overproduced and purified PerA and PerB from E. coli O157:H7 in E. coli BL21. The recombinant proteins were partly characterized and the final product of the reaction catalyzed by PerB was shown to be GDP-N-acetyl-d-perosamine by chromatography, mass spectrometry, and 1H-NMR. The functional expression of PerB provides another enzymatically defined pathway for the synthesis of GDP-deoxyhexoses, which is needed to further study the corresponding glycosyltransferases in vitro. 相似文献
9.
To assess treatment efficacy in clinical trials, certain clinicaloutcomes are repeatedly measured over time for the same subject.The difference in their means may characterize a treatment effect.Since treatment effectiveness lag and saturation times may exist,erosion of treatment effect often occurs during the observationperiod. Instead of using models based on ad hoc parametric orpurely nonparametric time-varying coefficients, we model thetreatment effectiveness durations, which are the time intervalsbetween the lag and saturation times. Then we use some meanresponse models to include such treatment effectiveness durations.Our methodology is demonstrated by simulations and analysisof a landmark HIV/AIDS clinical trial of short-course nevirapineagainst mother-to-child HIV vertical transmission during labourand delivery. 相似文献
10.
Cocoa breeders and growers continue to face the problem of high heterogeneity between individuals derived from one progeny. Vegetative propagation by somatic embryogenesis could be a way to increase genetic gains in the field. Somatic embryogenesis in cocoa is difficult and this species is considered as recalcitrant. This study was conducted to investigate the phenolic composition of cocoa flowers (the explants used to achieve somatic embryogenesis) and how it changes during the process, by means of histochemistry and conventional chemical techniques. In flowers, all parts contained polyphenolics but their locations were specific to the organ considered. After placing floral explants in vitro, the polyphenolic content was qualitatively modified and maintained in the calli throughout the culture process. Among the new polyphenolics, the three most abundant were isolated and characterized by 1H- and 13C-NMR. They were hydroxycinnamic acid amides: N-trans-caffeoyl-l-DOPA or clovamide, N-trans-p-coumaroyl-l-tyrosine or deoxiclovamide, and N-trans-caffeoyl-l-tyrosine. The same compounds were found also in fresh, unfermented cocoa beans. The synthesis kinetics for these compounds in calli, under different somatic embryogenesis conditions, revealed a higher concentration under non-embryogenic conditions. Given the antioxidant nature of these compounds, they could reflect the stress status of the tissues. 相似文献
11.
Y Yoshida Y Nakano T Nezu Y Yamashita T Koga 《The Journal of biological chemistry》1999,274(24):16933-16939
The serotype-specific polysaccharide antigen of Actinobacillus actinomycetemcomitans Y4 (serotype b) consists of D-fucose and L-rhamnose. Thymidine diphosphate (dTDP)-D-fucose is the activated nucleotide sugar form of D-fucose, which has been identified as a constituent of structural polysaccharides in only a few bacteria. In this paper, we show that three dTDP-D-fucose synthetic enzymes are encoded by genes in the gene cluster responsible for the synthesis of serotype b-specific polysaccharide in A. actinomycetemcomitans. The first and second steps of the dTDP-D-fucose synthetic pathway are catalyzed by D-glucose-1-phosphate thymidylyltransferase and dTDP-D-glucose 4,6-dehydratase, which are encoded by rmlA and rmlB in the gene cluster, respectively. These two reactions are common to the well studied dTDP-L-rhamnose synthetic pathway. However, the enzyme catalyzing the last step of the dTDP-D-fucose synthetic pathway has never been reported. We identified the fcd gene encoding a dTDP-4-keto-6-deoxy-D-glucose reductase. After purifying the three enzymes, their enzymatic activities were analyzed by reversed-phase high performance liquid chromatography. In addition, nuclear magnetic resonance analysis and gas-liquid chromatography analysis proved that the fcd gene product converts dTDP-4-keto-6-deoxy-D-glucose to dTDP-D-fucose. Moreover, kinetic analysis of the enzyme indicated that the Km values for dTDP-4-keto-6-deoxy-D-glucose and NADPH are 97.3 and 28.7 microM, respectively, and that the enzyme follows the sequential mechanism. This paper is the first report on the dTDP-D-fucose synthetic pathway and dTDP-4-keto-6-deoxy-D-glucose reductase. 相似文献
12.
The 4-amino-6-deoxy-monosaccharide D-perosamine is an important element in the glycosylation of interesting cell products, such as antibiotics and lipopolysaccharides (LPS) of Gram-positive and Gram-negative bacteria. The biosynthetic pathway of the precursor molecule, GDP-D-perosamine, in Vibrio cholerae O1 starts with an isomerisation of fructose-6-phosphate catalyzed by the bifunctional enzyme phosphomannose isomerase-guanosine diphosphomannose pyrophosphorylase (RfbA; E.C. 2.7.7.22) creating the intermediate mannose-6-phosphate, which is subsequently converted by the phosphomanno-mutase (RfbB; E.C. 5.4.2.8) and further by RfbA to GDP-D-mannose, to GDP-4 keto-6-deoxymannose by a 4,6-dehydratase (RfbD; E.C. 4.2.1.47) and finally to GDP-D-perosamine by an aminotransferase (RfbE; E.C. not yet classified). We cloned the rfbD and the rfbE genes of V. cholerae O1 in Escherichia coli expression vectors. Both biosynthetic enzymes were overproduced in E. coli BL21 (DE3) and their activities were analyzed. The enzymatic conversion from GDP-D-mannose to GDP-D-perosamine was optimized and the final product, GDP-D-perosamine, was purified and identified by nuclear magnetic resonance, mass spectrometry, and chromatography. The catalytically active form of the GDP-4-keto-6-deoxy-D-mannose-4-aminotransferase seems to be a tetramer of 170 kDa. The His-tag RfbE fusion protein has a Km of 0.06 mM and a Vmax value of 38 nkat/mg protein for the substrate GDP-4-keto-6-deoxy-D-mannose. The Km and Vmax values for the cosubstrate L-glutamate were 0.1 mM and 42 nkat/mg protein, respectively. The intention of this work is to establish a basis for both the in vitro production of GDP-D-perosamine and for an in vivo perosaminylation system in a suitable bacterial host, preferably E. coli. 相似文献
13.
An epimerase-reductase in L-fucose synthesis 总被引:2,自引:0,他引:2
The first committed enzyme in GDP-L-fucose formation from GDP-D-mannose is GDP-D-mannose 4,6-dehydratase, which forms GDP-4-keto-6-deoxy-D-mannose. The uncertain enzymatic steps beyond this point were examined in this study. Assays were developed for the epimerase and reductase activities which the putative pathway would predict. A protein was isolated exhibiting homogeneity by several criteria. This single protein, which forms GDP-L-fucose from GDP-4-keto-6-deoxy-D-mannose and NADH, appears to possess both epimerase and reductase capabilities and may be termed GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase. Analysis on a molecular sieve column using fast protein liquid chromatography established a molecular weight of 63,100 for the native enzyme, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis established a subunit molecular weight of 31,500. 相似文献
14.
L-Colitose is a 3,6-dideoxyhexose found in the O-antigen of Gram-negative lipopolysaccharides. To study the biosynthesis of this unusual sugar, we have cloned and sequenced the L-colitose biosynthetic gene cluster from Yersinia pseudotuberculosis VI. The colD and colC genes in this cluster have been overexpressed and each gene product has been purified and characterized. Our results showed that ColD functions as GDP-4-keto-6-deoxy-D-mannose-3-dehydrase responsible for C-3 deoxygenation of GDP-4-keto-6-deoxy-D-mannose. This enzyme is coenzyme B(6)-dependent and its catalysis is initiated by a transamination step in which pyridoxal 5'-phosphate (PLP) is converted to pyridoxamine 5'-phosphate (PMP) in the presene of L-glutamate. This coenzyme forms a Schiff base with the keto sugar substrate and the resulting adduct undergoes a PMP-mediated beta-dehydration reaction to give a sugar enamine intermediate, which after tautomerization and hydrolysis to release ammonia yields GDP-4-keto-3,6-dideoxy-D-mannose as the product. The combined transamination-deoxygenation activity places ColD in a class by itself. Our studies also established ColC as GDP-L-colitose synthase, which is a bifunctional enzyme catalyzing the C-5 epimerization of GDP-4-keto-3,6-dideoxy-D-mannose and the subsequent C-4 keto reduction of the resulting L-epimer to give GDP-L-colitose. Reported herein are the detailed accounts of the overexpression, purification, and characterization of ColD and ColC. Our studies show that their modes of action in the biosynthesis of GDP-L-colitose represent a new deoxygenation paradigm in deoxysugar biosynthesis. 相似文献
15.
Perosamine (4-amino-4,6-dideoxy- d-mannose), or its N-acetylated form, is one of several dideoxy sugars found in the O-antigens of such infamous Gram-negative bacteria as Vibrio cholerae O1 and Escherichia coli O157:H7. It is added to the bacterial O-antigen via a nucleotide-linked version, namely GDP-perosamine. Three enzymes are required for the biosynthesis of GDP-perosamine starting from mannose 1-phosphate. The focus of this investigation is GDP-perosamine synthase from Caulobacter crescentus, which catalyzes the final step in GDP-perosamine synthesis, the conversion of GDP-4-keto-6-deoxymannose to GDP-perosamine. The enzyme is PLP-dependent and belongs to the aspartate aminotransferase superfamily. It contains the typically conserved active site lysine residue, which forms a Schiff base with the PLP cofactor. Two crystal structures were determined for this investigation: a site-directed mutant protein (K186A) complexed with GDP-perosamine and the wild-type enzyme complexed with an unnatural ligand, GDP-3-deoxyperosamine. These structures, determined to 1.6 and 1.7 A resolution, respectively, revealed the manner in which products, and presumably substrates, are accommodated within the active site pocket of GDP-perosamine synthase. Additional kinetic analyses using both the natural and unnatural substrates revealed that the K m for the unnatural substrate was unperturbed relative to that of the natural substrate, but the k cat was lowered by a factor of approximately 200. Taken together, these studies shed light on why GDP-perosamine synthase functions as an aminotransferase whereas another very similar PLP-dependent enzyme, GDP-4-keto-6-deoxy- d-mannose 3-dehydratase or ColD, catalyzes a dehydration reaction using the same substrate. 相似文献
16.
Surfactant protein D (SP-D), one of the members of the collectin family of C-type lectins, is an important component of pulmonary innate immunity. SP-D binds carbohydrates in a calcium-dependent manner, but the mechanisms governing its ligand recognition specificity are not well understood. SP-D binds glucose (Glc) stronger than N-acetylglucosamine (GlcNAc). Structural superimposition of hSP-D with mannose- binding protein C (MBP-C) complexed with GlcNAc reveals steric clashes between the ligand and the side chain of Arg343 in hSP-D. To test whether Arg343 contributes to Glc > GlcNAc recognition specificity, we constructed a computational model of Arg343-->Val (R343V) mutant hSP-D based on homology with MBP-C. Automated docking of alpha-Me-Glc and alpha-Me-GlcNAc into wild-type hSP-D and the R343V mutant of hSP-D suggests that Arg343 is critical in determining ligand-binding specificity by sterically prohibiting one binding orientation. To empirically test the docking predictions, an R343V mutant recombinant hSP-D was constructed. Inhibition analysis shows that the R343V mutant binds both Glc and GlcNAc with higher affinity than the wild-type protein and that the R343V mutant binds Glc and GlcNAc equally well. These data demonstrate that Arg343 is critical for hSP-D recognition specificity and plays a key role in defining ligand specificity differences between MBP and SP-D. Additionally, our results suggest that the number of binding orientations contributes to monosaccharide binding affinity. 相似文献
17.
Di- and trideoxysugars are an important class of carbohydrates synthesized by certain plants, fungi, and bacteria. Colitose, for example, is a 3,6-dideoxysugar found in the O-antigens of Gram-negative bacteria such as Escherichia coli, Salmonella enterica, Yersinia pseudotuberculosis, and Vibrio cholerae, among others. These types of dideoxysugars are thought to serve as antigenic determinants and to play key roles in bacterial defense and survival. Four enzymes are required for the biochemical synthesis of colitose starting from mannose-1-phosphate. The focus of this investigation, GDP-4-keto-6-deoxy-d-mannose-3-dehydratase (ColD), catalyzes the third step in the pathway, namely the PLP-dependent removal of the C3'-hydroxyl group from GDP-4-keto-6-deoxymannose. Whereas most PLP-dependent enzymes contain an active site lysine, ColD utilizes a histidine as its catalytic acid/base. The ping-pong mechanism of the enzyme first involves the conversion of PLP to PMP followed by the dehydration step. Here we present the three-dimensional structure of a site-directed mutant form of ColD whereby the active site histidine has been replaced with a lysine. The electron density reveals that the geminal diamine, a tetrahedral intermediate in the formation of PMP from PLP, has been trapped within the active site region. Functional assays further demonstrate that this mutant form of ColD cannot catalyze the dehydration reaction. 相似文献
18.
Cook PD Thoden JB Holden HM 《Protein science : a publication of the Protein Society》2006,15(9):2093-2106
L-colitose is a 3,6-dideoxysugar found in the O-antigens of some Gram-negative bacteria such as Escherichia coli and in marine bacteria such as Pseudoalteromonas tetraodonis. The focus of this investigation, GDP-4-keto-6-deoxy-D-mannose-3-dehydratase, catalyzes the third step in colitose production, which is the removal of the hydroxyl group at C3' of GDP-4-keto-6-deoxymannose. It is an especially intriguing PLP-dependent enzyme in that it acts as both a transaminase and a dehydratase. Here we present the first X-ray structure of this enzyme isolated from E. coli Strain 5a, type O55:H7. The two subunits of the protein form a tight dimer with a buried surface area of approximately 5000 A2. This is a characteristic feature of the aspartate aminotransferase superfamily. Although the PLP-binding pocket is formed primarily by one subunit, there is a loop, delineated by Phe 240 to Glu 253 in the second subunit, that completes the active site architecture. The hydrated form of PLP was observed in one of the enzyme/cofactor complexes described here. Amino acid residues involved in anchoring the cofactor to the protein include Gly 56, Ser 57, Asp 159, Glu 162, and Ser 183 from one subunit and Asn 248 from the second monomer. In the second enzyme/cofactor complex reported, a glutamate ketimine intermediate was found trapped in the active site. Taken together, these two structures, along with previously reported biochemical data, support the role of His 188 as the active site base required for catalysis. 相似文献
19.
We consider a unified least absolute deviation estimator forstationary and nonstationary fractionally integrated autoregressivemoving average models with conditional heteroscedasticity. Itsasymptotic normality is established when the second momentsof errors and innovations are finite. Several other alternativeestimators are also discussed and are shown to be less efficientand less robust than the proposed approach. A diagnostic tool,consisting of two portmanteau tests, is designed to check whetheror not the estimated models are adequate. The simulation experimentsgive further support to our model and the results for the absolutereturns of the Dow Jones Industrial Average Index daily closingprice demonstrate their usefulness in modelling time seriesexhibiting the features of long memory, conditional heteroscedasticityand heavy tails. 相似文献
20.
Yan Ren Haiyan Wang Yuriy A. Knirel Wei Chen 《Biochemical and biophysical research communications》2010,391(4):1663-5984
Mortierella alpina is a filamentous fungus commonly found in soil, which is able to produce large amount of polyunsaturated fatty acids. l-Fucose is an important sugar found in a diverse range of organisms, playing a variety of biological roles. In this study, we characterized the de novo biosynthetic pathway of GDP-l-fucose (the nucleotide-activated form of l-fucose) in M. alpina. Genes encoding GDP-d-mannose 4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose 3,5-epimerase/4-reductase (GMER) were expressed heterologously in Escherichia coli. The recombinant enzymes were produced as His-tagged fusion proteins. Conversion of GDP-mannose to GDP-4-keto-6-deoxy mannose by GMD and GDP-4-keto-6-deoxy mannose to GDP-l-fucose by GMER were analyzed by capillary electrophoresis, electro-spray ionization-mass spectrometry, and nuclear magnetic resonance spectroscopy. The km values of GMD for GDP-mannose and GMER for GDP-4-keto-6-deoxy mannose were determined to be 0.77 mM and 1.047 mM, respectively. Both NADH and NADPH may be used by GMER as the coenzyme. The optimum temperature and pH were determined to be 37 °C and pH 9.0 (GMD) or pH 7.0 (GMER). Divalent cations are not required for GMD and GMER activity, and the activities of both enzymes may be enhanced by DTT. To our knowledge this is the first report on the characterization of GDP-l-fucose biosynthetic pathway in fungi. 相似文献