Disruption of a DUF247 Containing Protein Alters Cell Wall Polysaccharides and Reduces Growth in .

Plant cell wall biosynthesis is a complex process that requires proteins and enzymes from glycan synthesis to wall assembly. We show that disruption of (), a member of the multigene family containing 28 genes in , results in alterations to the structure and composition of cell wall polysaccharides and reduced growth and plant size. An ELISA using cell wall antibodies shows that the mutants also exhibit ~50% reductions in xyloglucan (XyG), glucuronoxylan (GX) and heteromannan (HM) epitopes in the NaOH fraction and ~50% increases in homogalacturonan (HG) epitopes in the CDTA fraction.

Crystal structure and identification of amino acid residues for catalysis and binding of GH3 AnBX β-xylosidase from Aspergillus niger.

β-Xylosidases catalyze the hydrolysis of xylooligosaccharides to xylose in the final step of hemicellulose degradation. AnBX, which is a GH3 β-xylosidase from Aspergillus niger, has a high catalytic efficiency toward xyloside substrates. In this study, we report the three-dimensional structure and the identification of catalytic and substrate binding residues of AnBX by performing site-directed mutagenesis, kinetic analysis, and NMR spectroscopy-associated analysis of the azide rescue reaction.

Anthranilic Acid Accumulation in Saccharomyces cerevisiae Induced by Expression of a Nonribosomal Peptide Synthetase Gene from Paecilomyces cinnamomeus BCC 9616.

Heterologous expression of nrps33, a nonribosomal peptide synthetase gene, from Paecilomyces cinnamomeus BCC 9616 in Saccharomyces cerevisiae unexpectedly resulted in the accumulation of anthranilic acid, an intermediate in tryptophan biosynthesis. Based on transcriptomic and real-time quantitative polymerase chain reaction (RT-qPCR) results, expression of nrps33 affected the transcription of tryptophan biosynthesis genes especially TRP1 which is also the selectable auxotrophic marker for the expression vector used in this work. The product of nrps33 could inhibit the activity of Trp4 involved in the conversion of anthranilate to N-(5'-phosphoribosyl)anthranilate and therefore caused the accumulation of anthranilic acid.

-Methylation controls the biosynthetic programming of alternapyrone.

Alternapyrone is a highly methylated polyene α-pyrone biosynthesised by a highly reducing polyketide synthase. Mutations of the catalytic dyad residues, H1578A/Q and E1604A, of the -methyltransferase domain resulted in either significantly reduced or no production of alternapyrone, indicating the importance of -methylation for alternapyrone biosynthesis.

Maillard reaction products-based encapsulant system formed between chitosan and corn syrup solids: Influence of solution pH on formation kinetic and antioxidant activity.

Maillard reaction products (MRPs) between chitosan and various sugars with enhanced antioxidant activity were previously produced. However, few reports address the chitosan and corn syrup solids system that has been successfully used to encapsulate nutraceutical oils. Maillard reaction is pH-responsive, the influence of solution pH on the formation kinetic and antioxidant activity of MRPs was therefore evaluated in this work.

Absolute configuration of azaphilones from KB9 and solvent effects on their keto and enol forms.

fermented rice, also known as red yeast rice, exhibits a broad spectrum of biological activities due to its chemical constituents, such as monacolins and azaphilone pigments. Here, we cultured KB9 in a liquid malt medium instead of on rice as a carbon source. Eleven known compounds (-) containing azaphilones and their early intermediate were isolated and identified.

Combination of H and C NMR for quantitative analysis of the orange pigments produced by KB9.

Two orange pigments, rubropunctatin () and monascorubrin (), along with the yellow pigments, monascin () and ankaflavin (), were isolated from KB9-fermented rice, also known as red yeast rice. The orange pigments exhibit a broad spectrum of biological activities and appeared to be the major components of this fermented rice. In this work, quantitative H NMR (qHNMR) and C NMR experiments were used to determine the amounts of the two orange pigments in a crude extract in which most of the H NMR signals of the two compounds were indistinguishable.

Solution Structure and Conformational Dynamics of a Doublet Acyl Carrier Protein from Prodigiosin Biosynthesis.

The acyl carrier protein (ACP) is an indispensable component of both fatty acid and polyketide synthases and is primarily responsible for delivering acyl intermediates to enzymatic partners. At present, increasing numbers of multidomain ACPs have been discovered with roles in molecular recognition of trans-acting enzymatic partners as well as increasing metabolic flux. Further structural information is required to provide insight into their function, yet to date, the only high-resolution structure of this class to be determined is that of the doublet ACP (two continuous ACP domains) from mupirocin synthase.

SAXS reveals highly flexible interdomain linkers of tandem acyl carrier protein-thioesterase domains from a fungal nonreducing polyketide synthase.

Menisporopsin A is a fungal bioactive macrocyclic polylactone, the biosynthesis of which requires only reducing (R) and nonreducing (NR) polyketide synthases (PKSs) to guide a series of esterification and cyclolactonization reactions. There is no structural information pertaining to these PKSs. Here, we report the solution characterization of singlet and doublet acyl carrier protein (ACP and ACP -ACP )-thioesterase (TE) domains from NR-PKS involved in menisporopsin A biosynthesis.

Synthesis of long-chain alkyl glucosides via reverse hydrolysis reactions catalyzed by an engineered β-glucosidase.

Long-chain alkyl glucosides, such as octyl and decyl β-d-glucopyranosides (OG and DG, respectively), are regarded as a new generation of biodegradable, non-ionic surfactants. Previously, the mutants of Dalbergia cochinchinensis Pierre dalcochinase showed potential in the synthesis of oligosaccharides and alkyl glucosides. In this study, the N189F dalcochinase mutant gave the highest yields of OG and DG synthesis under reverse hydrolysis conditions.

Improved synthesis of long-chain alkyl glucosides catalyzed by an engineered β-glucosidase in organic solvents and ionic liquids.

Objective: To synthesize octyl β-D-glucopyranoside (OG) and decyl β-D-glucopyranoside (DG) in three non-aqueous reaction systems, namely organic solvents, ionic liquids and co-solvent mixtures, via reverse hydrolysis reactions catalyzed by the N189F dalcochinase mutant.

Results: The highest yield of OG (67 mol%) was obtained in the reaction containing 0.5 M glucose, 3 unit ml enzyme in 20% (v/v) octanol and 70% (v/v) [BMIm][PF] at 30 °C.

Sarpeptins A and B, Lipopeptides Produced by sp. KO-7888 Overexpressing a Specific SARP Regulator.

Whole genome analysis of sp. KO-7888 has revealed various pathway-specific transcriptional regulatory genes associated with silent biosynthetic gene clusters. A antibiotic regulatory protein gene, , located adjacent to a novel nonribosomal peptide synthetase (NRPS) gene cluster, was overexpressed in the wild-type strain.

Heterologous biosynthesis of a fungal macrocyclic polylactone requires only two iterative polyketide synthases.

Menisporopsin A is a bioactive macrocyclic polylactone produced by the fungus Menisporopsis theobromae BCC 4162. A scheme for the biosynthesis of this compound has been proposed, in which reducing (R) and non-reducing (NR) polyketide synthases (PKSs) would catalyze the formation of each menisporopsin A subunit, while an additional non-ribosomal peptide synthetase (NRPS)-like enzyme would be required to perform multiple esterification and cyclolactonization reactions. Transcriptome analysis of M.

Molecular Characterization and Potential Synthetic Applications of GH1 β-Glucosidase from Higher Termite Microcerotermes annandalei.

A novel β-glucosidase from higher termite Microcerotermes annandalei (MaBG) was obtained via a screening method targeting β-glucosidases with increased activities in the presence of glucose. The purified natural MaBG showed a subunit molecular weight of 55 kDa and existed in a native form as a dimer without any glycosylation. Gene-specific primers designed from its partial amino acid sequences were used to amplify the corresponding 1,419-bp coding sequence of MaBG which encodes a 472-amino acid glycoside hydrolase family 1 (GH1) β-glucosidase.

Structural and Functional Studies of the Daunorubicin Priming Ketosynthase DpsC.

Daunorubicin is a type II polyketide, one of a large class of polyaromatic natural products with anticancer, antibiotic, and antiviral activity. Type II polyketides are formed by the assembly of malonyl-CoA building blocks, though in rare cases, biosynthesis is initiated by the incorporation of a nonmalonyl derived starter unit, which adds molecular diversity to the poly-β-ketone backbone. Priming mechanisms for the transfer of novel starter units onto polyketide synthases (PKS) are still poorly understood.

Biochemical characterization of plasmepsin V from Plasmodium vivax Thailand isolates: Substrate specificity and enzyme inhibition.

Plasmepsin V (PMV) is a Plasmodium aspartic protease responsible for the cleavage of the Plasmodium export element (PEXEL) motif, which is an essential step for export of PEXEL containing proteins and crucial for parasite viability. Here we describe the genetic polymorphism of Plasmodium vivax PMV (PvPMV) Thailand isolates, followed by cloning, expression, purification and characterization of PvPMV-Thai, presenting the pro- and mature-form of PvPMV-Thai. With our refolding and purification method, approximately 1mg of PvPMV-Thai was obtained from 1g of washed inclusion bodies.

Antibacterial activity of cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr) from Streptomyces sp. strain 22-4 against phytopathogenic bacteria.

Two bioactive cyclic dipeptides, cyclo(L-Pro-L-Tyr) and cyclo(D-Pro-L-Tyr), were isolated from the culture broth of Streptomyces sp. strain 22-4 and tested against three economically important plant pathogens, Xanthomonas axonopodis pv. citri, Ralstonia solanacearum and Clavibacter michiganensis.

Cloning, expression and characterization of β-xylosidase from Aspergillus niger ASKU28.

β-Xylosidases catalyze the breakdown of β-1,4-xylooligosaccharides, which are produced from degradation of xylan by xylanases, to fermentable xylose. Due to their important role in xylan degradation, there is an interest in using these enzymes in biofuel production from lignocellulosic biomass. In this study, the coding sequence of a glycoside hydrolase family 3 β-xylosidase from Aspergillus niger ASKU28 (AnBX) was cloned and expressed in Pichia pastoris as an N-terminal fusion protein with the α-mating factor signal sequence (α-MF) and a poly-histidine tag.

A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis.

Type I polyketide synthases often use programmed β-branching, via enzymes of a 'hydroxymethylglutaryl-CoA synthase (HCS) cassette', to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in acyl carrier proteins (ACPs) where β-branching is known to occur. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity.

Biosynthetic origins of menisporopsin A.

Menisporopsin A, produced by Menisporopsis theobromae, shows antimalarial, antimycobacterial, and cytotoxic activities. Here, we report the first (13)C incorporations at individual carbons of menisporopsin A using sodium [1-(13)C] and [2-(13)C] acetate. This result indicates that each of the subunits of the pentalactone menisporopsin A is assembled by a polyketide synthase.

Overproduction of the N-terminal anticodon-binding domain of the non-discriminating aspartyl-tRNA synthetase from Helicobacter pylori for crystallization and NMR measurements.

Aminoacyl-tRNA synthetases (aaRSs) covalently attach an amino acid to its cognate tRNA isoacceptors through an ester bond. The standard set of 20 amino acids implies 20 aaRSs for each pair of amino acid/tRNA isoacceptors. However, the genomes of all archaea and some bacteria do not encode for a complete set of 20 aaRSs.

An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution.

Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2.

Recognition of intermediate functionality by acyl carrier protein over a complete cycle of fatty acid biosynthesis.

It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR.

Solution structure of an acyl carrier protein domain from a fungal type I polyketide synthase.

Acyl (peptidyl) carrier protein (ACP or PCP) is a crucial component involved in the transfer of thiol ester-bound intermediates during the biosynthesis of primary and secondary metabolites such as fatty acids, polyketides, and nonribosomal peptides. Although many carrier protein three-dimensional structures have been determined, to date there is no model available for a fungal type I polyketide synthase ACP. Here we report the solution structure of the norsolorinic acid synthase (NSAS) holo ACP domain that has been excised from the full-length multifunctional enzyme.