Structure of the oligosaccharides isolated from Dalbergia sissoo Roxb. leaf polysaccharide

A water-soluble polysaccharide isolated from Dalbergia sissoo Roxb. leaves was purified and major homogeneous fraction obtained by GPC. Complete hydrolysis of the polysaccharide followed by paper chromatography and GLC analysis indicated the presence of l-rhamnose, d-glucuronic acid, d-galactose and d-glucose in molar ratio of 1:1:2:2.33, respectively. Partial hydrolysis of the polysaccharide furnished one tri-[I], one hepta-[II] and one nona-[III] saccharides. Hydrolysis of the oligosaccharide I, II and III followed by GLC analysis furnished d-glucose and l-rhamnose (2:1); l-rhamnose, d-galactose and d-glucuronic acid (1:3:3); and l-rhamnose, d-galactose and d-glucose (1:3:5), respectively. Methylation analysis and periodate oxidation of the oligosaccharide I indicated the presence of two non reducing glucose units linked to rhamnose by 1→2 and 1→4 linkages, respectively. Oligosaccharide II is a branched molecule with a main chain consisting of 1,3-linked β-d-galactopyranosyl (2 mol), 1,3,4 linked α-l-rhamnopyranosyl (1 mol) and 1,4,6 linked β-d-galactopyranosyl unit (1 mol) and non reducing β-d-glucuronic acid at the end along with side chains of β-d-glucouronopyranosyl units (2 mol). Oligosaccharide III is also a branched molecule with a main chain consisting of 1,3,4 linked α-l-rhamnopyranosyl (1 mol), 1,2,4 linked β-d-glucopyranosyl (1 mol), 1,3 and 1,4 linked β-d-galactopyranosyl (2 and 1 mol, respectively) having β-d-glucopyranosyl as a non reducing end.     

The biosynthetic pathway of crucifer phytoalexins and phytoanticipins: De novo incorporation of deuterated tryptophans and quasi-natural compounds

Although several biosynthetic intermediates in pathways to cruciferous phytoalexins and phytoanticipins are common, questions regarding the introduction of substituents at N-1 of the indole moiety remain unanswered. Toward this end, we investigated the potential incorporations of several perdeuterated d- and l-1′-methoxytryptophans, d- and l-tryptophans and other indol-3-yl derivatives into pertinent phytoalexins and phytoanticipins (indolyl glucosinolates) produced in rutabaga (Brassica napus L. ssp. rapifera) roots. In addition, we probed the potential transformations of quasi-natural compounds, these being analogues of biosynthetic intermediates that might lead to “quasi-natural” products (products similar to natural products but not produced under natural conditions). No detectable incorporations of deuterium labeled 1′-methoxytryptophans into phytoalexins or glucobrassicin were detected. l-tryptophan was incorporated in a higher percentage than d-tryptophan into both phytoalexins and phytoanticipins. However, in the case of the phytoalexin rapalexin A, both d- and l-tryptophan were incorporated to the same extent. Furthermore, the transformations of both 1′-methylindolyl-3′-acetaldoxime and 1′-methylindolyl-3′-acetothiohydroxamic acid (quasi-natural products) into 1′-methylglucobrassicin but not into phytoalexins suggested that post-aldoxime enzymes in the biosynthetic pathway of indolyl glucosinolates are not substrate-specific. Hence, it would appear that the 1-methoxy substituent of the indole moiety is introduced downstream from tryptophan and that the post-aldoxime enzymes of the glucosinolate pathway are different from the enzymes of the phytoalexin pathway. A higher substrate specificity of some enzymes of the phytoalexin pathway might explain the relatively lower structural diversity among phytoalexins than among glucosinolates.     

Identification of two novel xylanase-encoding genes (xyn5 and xyn6) from Acrophialophora nainiana and heterologous expression of xyn6 in Trichoderma reesei

Two novel genes, xyn5 and xyn6, coding for family 11 xylanases, were isolated from the thermotolerant filamentous fungus, Acrophialophora nainiana, by PCR using degenerate primers. The xyn6 gene was further expressed in Trichoderma reesei. DNA sequence analysis of xyn6 revealed an open reading frame (ORF) of 708 bp, interrupted by an intron of 58 bp. The xyn6 ORF encodes a predicted protein of 236 amino acid residues. The mature recombinant XynVI protein had a molecular mass of about 19 kDa, as estimated by gel electrophoresis. Analysis of the predicted amino acid sequence of XynVI paves the way for rational protein engineering by site-directed mutagenesis aiming to increase the thermostability of the heterologously-expressed xylanase.     

Glycosylated nervogenic acid derivatives from Liparis condylobulbon (Reichb.f.) leaves

Three new nervogenic acid glycosides, 1-O-α-l-rhamnopyranosyl 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoate, 3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoic acid, and bis{3,5-bis(3-methyl-but-2-enyl)-4-O-[α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl]-benzoyl} 1,2-O-β-d-glucopyranose, which we named condobulbosides A–C, were isolated from a methanol extract of the leaves of Liparis condylobulbon together with an apigenin C-glycoside, schaftoside. Their structures were established on the basis of spectral techniques, namely, UV, IR, HR-MS spectroscopy, both 1D and 2D NMR experiments, and chemical reactions.     

Gene cloning, purification, and characterization of α-methylserine aldolase from Bosea sp. AJ110407 and its applicability for the enzymatic synthesis of α-methyl-l-serine and α-ethyl-l-serine

The gene encoding α-methylserine aldolase was isolated from Bosea sp. AJ110407. Sequence analysis revealed that the predicted amino acid sequence encoded by the 1320-bp open reading frame was 65.0% similar to the corresponding sequence of the enzyme isolated from Ralstonia sp. AJ110405. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. Gel filtration revealed the molecular mass of the purified enzyme to be approximately 78 kDa, suggesting that the enzyme is a homodimer. The enzyme exhibited a specific peak at 429 nm in the spectrum and contained 1 mol pyridoxal 5′-phosphate per mole of the subunit. The V_max value was 1.40 μmol min⁻¹ mg⁻¹, and the K_m value was 1.5 mM for the reaction wherein formaldehyde was released from α-methyl-l-serine. This enzyme could also catalyze the reverse reaction, i.e., the synthesis of α-methyl-l-serine from l-alanine and formaldehyde. This activity was inhibited in the excess of formaldehyde; however, α-methyl-l-serine was efficiently produced from l-alanine in the presence of formaldehyde. This method was also applicable for producing α-ethyl-l-serine from l-2-aminobutyric acid.     

Functional analysis of the <Emphasis Type="Italic">egl3</Emphasis> upstream region in filamentous fungus <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

In the filamentous fungus Trichoderma reesei, endoglucanase III (EGIII) is coordinately expressed with other cellulases during growth on cellulose, its derivatives, and L-sorbose. To elucidate EGIII induction mechanism, we cloned and sequenced the upstream region of egl3 encoding EGIII. Two GGCTAA motifs, a putative binding site for ACEII and xylanase regulator Xyr1, were found on the template strand of the egl3 upstream region. Deletion analysis of the egl3 upstream region using the beta-glucuronidase (GUS) reporter system revealed that removal of regions containing the GGCTAA motifs and the region between −1,045 and −1,002 bp containing GGCTAT motif severely affected GUS inducibility. Furthermore, mutation of the two GGCTAA motifs and the GGCTAT motif of this region led to a significant decrease in GUS activity. These data indicate that both GGCTAA and GGCTAT are key motifs for egl3 expression, and that egl3 induction may also be controlled by Xyr1. This hypothesis was supported by in vitro electrophoretic mobility shift assay, in which heterologously expressed Xyr1 specifically bound not only GGCTAA but also GGCTAT motif.     

Structural characterization of an O-linked tetrasaccharide from Pseudomonas syringae pv. tabaci flagellin

The flagellin of Pseudomonas syringae pv. tabaci is a glycoprotein that contains O-linked oligosaccharides composed of rhamnosyl and 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methylglucosyl residues. These O-linked glycans are released by hydrazinolysis and then labeled at their reducing ends with 2-aminopyridine (PA). A PA-labeled trisaccharide and a PA-labeled tetrasaccharide are isolated by normal-phase high-performance liquid chromatography. These oligosaccharides are structurally characterized using mass spectrometry and NMR spectroscopy. Our data show that P. syringae pv. tabaci flagellin is glycosylated with a tetrasaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap-(1→2)-α-l-Rha-(1→, as well a trisaccharide, 4,6-dideoxy-4-(3-hydroxybutanamido)-2-O-methyl-Glcp-(1→3)-α-l-Rhap-(1→2)-α-l-Rha-(1→, which was identified in a previous study.     

Sequence analysis of rice rubi3 promoter gene expression cassettes for improved transgene expression

In a construct containing a GUS reporter gene driven by the 5′ regulatory elements from rubi3, expression was enhanced 4-fold when a 20-nucleotide (nt) GUS 5′ untranslated sequence was replaced with 9 nt sequences derived from rubi3′s second exon. The roles of the sequences immediately upstream from the GUS translation initiation codon, and their significance in gene expression, were investigated. Sequence analysis suggests that complementarity between sequences immediately 5′ of a translation initiation codon and the rice 17S rRNA may be responsible for the reduction in protein levels from constructs containing the GUS leader sequence. The results demonstrate an affect sequences immediately upstream from transgenic coding sequences have on expression, and when using the rubi3 5′ regulatory sequence in particular.     

Increased Production of Xylanase by Expression of a Truncated Version of the xyn11A Gene from Nonomuraea flexuosa in Trichoderma reesei

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei. The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3′ to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 (cel7A, cbh1) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A. The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter⁻¹ of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.     

Hemiterpene glucosides and other constituents from Spiraea canescens

Five glycosides, 2-(trans-cinnamoyloxy-methyl)-1-butene-4-O-β-d-glucopyranoside (1), 4-(6′-O-trans-cinnamoyl)-(2-hydroxymethyl-4-hydroxy-butenyl-β-d-glucopyranoside (2), 6′′-O-trans-p-coumaroyl-(4-hydroxybenzoyl)-β-d-glucopyranoside (3), 6′-O-(4-methoxy-trans-cinnamoyl) α/β-d-glucopyranose (4) 6′-O-(4′′-methoxy-trans-cinnamoyl)-kaempferol-3-β-d-glucopyranoside (7) along with six known compounds, (+)-isolariciresinol 3a-O-β-d-glucopyranoside (8) (+)-lyoniresinol 3a-O-β-d-glucopyranoside (9), apigenin 7-O-β-d-glucopyranoside (10), quercetin 3-O-β-d-glucopyranoside (11), 6′-O-cinnamoyl-α/β-d-glucopyranose (6) 6’-O-p-coumaroyl-α/β-d-glucopyranose (5) were isolated from the whole plant of Spiraea canescens. Some of these compounds showed potent radical scavenging activity in relevant non-physiological assays. Their structures were determined by NMR spectroscopic and CID mass spectrometric techniques.     

A pyrroloquinoline quinine-dependent membrane-bound d-sorbitol dehydrogenase from Gluconobacter oxydans exhibits an ordered Bi Bi reaction mechanism

A membrane-bound pyrroloquinoline quinine (PQQ)-dependent d-sorbitol dehydrogenase (mSLDH) in Gluconobacter oxydans participates in the oxidation of d-sorbitol to l-sorbose by transferring electrons to ubiquinone which links to the respiratory chain. To elucidate the kinetic mechanism, the enzyme purified was subjected to two-substrate steady-state kinetic analysis, product and substrate inhibition studies. These kinetic data indicate that the catalytic reaction follows an ordered Bi Bi mechanism, where the substrates bind to the enzyme in a defined order (first ubiquinone followed by d-sorbitol), while products are released in sequence (first l-sorbose followed by ubiquinol). From these findings, we proposed that the native mSLDH bears two different substrate-binding sites, one for ubiquinone and the other for d-sorbitol, in addition to PQQ-binding and Mg²⁺-binding sites in the catalytic center.     

Hydroxylation of l-proline to cis-3-hydroxy-l-proline by recombinant Escherichia coli expressing a synthetic l-proline-3-hydroxylase gene

A synthetic gene encoding a Streptomyces l-proline-3-hydroxylase was constructed and used to produce the hydroxylase protein in recombinant Escherichia coli. A fermentation process for growth of this recombinant E. coli for enzyme production was scaled-up to 250 L. A biotransformation process was developed using cell suspensions of the recombinant E. coli and subsequently scaled-up to 10 L for conversion of l-proline to cis-3-hydroxy-l-proline. A reaction yield of 85 M% and d.e. of 99.9% was obtained for cis-3-hydroxy-l-proline.     

The O-specific polysaccharide structure and biosynthetic gene cluster of Yersinia pseudotuberculosis serotype O:11

In the Yersinia pseudotuberculosis serotyping scheme, 21 serotypes are present originating from about 30 different O-factors distributed within the species. With regard to the chemical structures of lipopolysaccharides (LPSs) and the genetic basis of their biosynthesis, a number, but not all, of Y. pseudotuberculosis strains representing different serotypes have been investigated. In order to present an overall picture of the relationship between genetics and structures, we have been working on the genetics and structures of various Y. pseudotuberculosis O-specific polysaccharides (OPSs). Here, we present a structural and genetic analysis of the Y. pseudotuberculosis serotype O:11 OPS. Our results showed that this OPS structure has the same backbone as that of Y. pseudotuberculosis O:1b, but with a 6d-l-Altf side-branch instead of Parf. The 3′ end of the gene cluster is the same as that for O:1b and has the genes for synthesis of the backbone and for processing the completed repeat unit. The 5′ end has genes for synthesis of 6d-l-Altf and its transfer to the repeating unit backbone. The pathway for the synthesis of the 6d-l-Altf appears to be different from that for 6d-l-Altp in Y. enterocolitica O:3. The chemical structure of the O:11 repeating unit is

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Production in <Emphasis Type="Italic">Trichoderma reesei</Emphasis> of three xylanases from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>: a recombinant thermoxylanase for biobleaching of kraft pulp

Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5–7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.