Plant Heat Shock Proteins Are More Effective in Enhancing Recombinant Alcohol Dehydrogenase Activity than Bacterial Ones .

Background: Recombinant proteins produced in the cell factories are used in biological research, pharmaceutical production, and biochemical and agricultural applications. Molecular chaperones, such as heat shock proteins (Hsps), are co-expressed with recombinant proteins to enhance their yield, stability, and activity. When () is used as a cell factory, Hsps are the frequently used co-expression partners.

Enhanced Acetate Tolerance and Recombinant Protein Accumulation in by Transgenic Expression of a Heat Shock Protein from Carrot ( L.).

Background: In () culture, acetate accumulates as an undesirable by-product of aerobic fermentation on glucose and inhibits cell growth and recombinant protein production.

Objectives: We examined whether the heterologous expression of a eukaryotic heat shock protein (Hsp) can confer tolerance to acetate in .

Materials And Methods: Transgenic cell lines (TCLs) heterologously expressing a small heat shock protein (sHsp) from carrot ( L.

Enhanced Production of Recombinant Alcohol Dehydrogenase Using the Genetically Engineered Escherichia coli Strain that Heterologously Expresses Carrot Heat Shock Protein 70.

Escherichia coli (E. coli) has been widely used as a host organism for producing recombinant proteins such as biocatalysts, antibody fragments, and therapeutic hormones. To enhance recombinant protein production, many E.

Crystal structures of an atypical aldehyde dehydrogenase having bidirectional oxidizing and reducing activities.

Aldehyde dehydrogenases (ALDHs) are NAD(P)-dependent oxidoreductases that catalyze the oxidation of a variety of aldehydes to their acid forms. In this study, we determined the crystal structures of ALDH from Bacillus cereus (BcALDH), alone, and in complex with NAD and NADP. This enzyme can oxidize all-trans-retinal to all-trans-retinoic acid using either NAD or NADP with equal efficiency, and atypically, as a minor activity, can reduce all-trans-retinal to all-trans-retinol using NADPH.

Heterologous Expression of the Carrot Hsp17.7 gene Increased Growth, Cell Viability, and Protein Solubility in Transformed Yeast (Saccharomyces cerevisiae) under Heat, Cold, Acid, and Osmotic Stress Conditions.

In industrial fermentation of yeast (Saccharomyces cerevisiae), culture conditions are often modified from the optimal growth conditions of the cells to maintain large-scale cultures and/or to increase recombinant protein production. However, altered growth conditions can be stressful to yeast cells resulting in reduced cell growth and viability. In this study, a small heat shock protein gene from carrot (Daucus carota L.

Catalytic Intermediate Crystal Structures of Cysteine Desulfurase from the Archaeon NA1.

NA1 is an anaerobic archaeon usually found in a deep-sea hydrothermal vent area, which can use elemental sulfur (S) as a terminal electron acceptor for energy. Sulfur, essential to many biomolecules such as sulfur-containing amino acids and cofactors including iron-sulfur cluster, is usually mobilized from cysteine by the pyridoxal 5'-phosphate- (PLP-) dependent enzyme of cysteine desulfurase (CDS). We determined the crystal structures of CDS from NA1 (ToCDS), which include native internal aldimine (NAT), gem-diamine (GD) with alanine, internal aldimine structure with existing alanine (IAA), and internal aldimine with persulfide-bound Cys356 (PSF) structures.

Crystallographic snapshots of active site metal shift in E. coli fructose 1,6-bisphosphate aldolase.

Fructose 1,6-bisphosphate aldolase (FBA) is important for both glycolysis and gluconeogenesis in life. Class II (zinc dependent) FBA is an attractive target for the development of antibiotics against protozoa, bacteria, and fungi, and is also widely used to produce various high-value stereoisomers in the chemical and pharmaceutical industry. In this study, the crystal structures of class II Escherichia coli FBA (EcFBA) were determined from four different crystals, with resolutions between 1.

Crystal Structures of Peptide Deformylase from Rice Pathogen Xanthomonas oryzae pv. oryzae in Complex with Substrate Peptides, Actinonin, and Fragment Chemical Compounds.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight on rice; this species is one of the most destructive pathogenic bacteria in rice cultivation worldwide. Peptide deformylase (PDF) catalyzes the removal of the N-formyl group from the N-terminus of newly synthesized polypeptides in bacterial cells and is an important target to develop antibacterial agents.

Structure of D-alanine-D-alanine ligase from Yersinia pestis: nucleotide phosphate recognition by the serine loop.

D-Alanyl-D-alanine is an essential precursor of bacterial peptidoglycan and is synthesized by D-alanine-D-alanine ligase (DDL) with hydrolysis of ATP; this reaction makes DDL an important drug target for the development of antibacterial agents. Five crystal structures of DDL from Yersinia pestis (YpDDL) were determined at 1.7-2.

The crystal structure of the D-alanine-D-alanine ligase from Acinetobacter baumannii suggests a flexible conformational change in the central domain before nucleotide binding.

Acinetobacter baumannii, which is emerging as a multidrug-resistant nosocomial pathogen, causes a number of diseases, including pneumonia, bacteremia, meningitis, and skin infections. With ATP hydrolysis, the D-alanine-D-alanine ligase (DDL) catalyzes the synthesis of D-alanyl-D-alanine, which is an essential component of bacterial peptidoglycan. In this study, we determined the crystal structure of DDL from A.

Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine β-lyase from Acinetobacter baumannii OXA-23.

Multidrug-resistant Acinetobacter baumannii (Ab) has emerged as a leading nosocomial pathogen because of its resistance to most currently available antibiotics. Cystathionine β-lyase (CBL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the second step in the transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids homocysteine and methionine. The enzymes of the transsulfuration pathway are considered to be attractive drug targets owing to their specificity to microbes and plants.

Crystallization and preliminary X-ray crystallographic analysis of the XoGroEL chaperonin from Xanthomonas oryzae pv. oryzae.

Along with the co-chaperonin GroES, the chaperonin GroEL plays an essential role in enhancing protein folding or refolding and in protecting proteins against misfolding and aggregation in the cellular environment. The XoGroEL gene (XOO_4288) from Xanthomonas oryzae pv. oryzae was cloned and the protein was expressed, purified and crystallized.

Divalent metal ion-based catalytic mechanism of the Nudix hydrolase Orf153 (YmfB) from Escherichia coli.

YmfB from Escherichia coli is the Nudix hydrolase involved in the metabolism of thiamine pyrophosphate, an important compound in primary metabolism and a cofactor of many enzymes. In addition, it hydrolyzes (d)NTPs to (d)NMPs and inorganic orthophosphates in a stepwise manner. The structures of YmfB alone and in complex with three sulfates and two manganese ions determined by X-ray crystallography, when compared with the structures of other Nudix hydrolases such as MutT, Ap4Aase and DR1025, provide insight into the unique hydrolysis mechanism of YmfB.

Crystal structures of d-alanine-d-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides.

D-Alanine-D-alanine ligase (DDL) catalyzes the biosynthesis of d-alanyl-d-alanine, an essential bacterial peptidoglycan precursor, and is an important drug target for the development of antibacterials. We determined four different crystal structures of DDL from Xanthomonas oryzae pv. oryzae (Xoo) causing Bacteria Blight (BB), which include apo, ADP-bound, ATP-bound, and AMPPNP-bound structures at the resolution between 2.

Crystal structure of XoLAP, a leucine aminopeptidase, from Xanthomonas oryzae pv. oryzae.

Aminopeptidases are metalloproteinases that degrade N-terminal residues from protein and play important roles in cell growth and development by controlling cell homeostasis and protein maturation. We determined the crystal structure of XoLAP, a leucyl aminopeptidase, at 2.6 Å resolution from Xanthomonas oryzae pv.

Expression, crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Campylobacter jejuni.

Campylobacter jejuni is one of the major foodborne pathogens causing human infection. Peptide deformylase, a metallohydrolase, catalyzes the deformylation of N-formylated methionine in newly synthesized polypeptides in prokaryotes and some eukaryotic organelles. The deformylation process is an essential step in protein synthesis and has attracted much attention as a potential target for the development of novel antibacterial agents.

Homologous expression and T3SS-dependent secretion of TAP-tagged Xo2276 in Xanthomonas oryzae pv. oryzae induced by rice leaf extract and its direct in vitro recognition of putative target DNA sequence.

Xo2276 is a putative transcription activator-like effector (TALE) in Xanthomonas oryzae pv. oryzae (Xoo). Xo2276 was expressed with a TAP-tag at the C-terminus in Xoo cells to enable quantitative analysis of protein expression and secretion.

Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine γ-synthase (XometB) from Xanthomonas oryzae pv. oryzae.

Cystathionine γ-synthase (CGS) catalyzes the first step in the transsulfuration pathway leading to the formation of cystathionine from O-succinylhomoserine and L-cysteine through a γ-replacement reaction. As an antibacterial drug target against Xanthomonas oryzae pv. oryzae (Xoo), CGS from Xoo (XometB) was cloned, expressed, purified and crystallized.

Crystallization and preliminary X-ray crystallographic analysis of β-ketoacyl-ACP synthase I (XoFabB) from Xanthomonas oryzae pv. oryzae.

The proteins in the fatty-acid synthesis pathway in bacteria have significant potential as targets for the development of antibacterial agents. An essential elongation step in fatty-acid synthesis is performed by β-ketoacyl-acyl carrier protein synthase I (FabB). The organism Xanthomonas oryzae pv.

Crystal structure of malonyl CoA-Acyl carrier protein transacylase from Xanthomanous oryzae pv. oryzae and its proposed binding with ACP.

Xanthomonas oryzae pv. oryzae (Xoo) is a plant bacterial pathogen that causes bacterial blight (BB) disease, resulting in serious production losses of rice. The crystal structure of malonyl CoA-acyl carrier protein transacylase (XoMCAT), encoded by the gene fabD (Xoo0880) from Xoo, was determined at 2.

DcHsp17.7, a small heat shock protein in carrot, is tissue-specifically expressed under salt stress and confers tolerance to salinity.

The expression and function of DcHsp17.7, a small heat shock protein in carrot (Daucus carota L.), were examined under salt stress, which is an exacerbating environmental condition due to water shortage and irrigation.

Crystal structure of Clostridium thermocellum ribose-5-phosphate isomerase B reveals properties critical for fast enzyme kinetics.

Ribose-5-phosphate isomerase (Rpi) catalyzes the conversion of D-ribose 5-phosphate (R5P) to D-ribulose 5-phosphate, which is an important step in the non-oxidative pathway of the pentose phosphate pathway and the Calvin cycle of photosynthesis. Recently, Rpis have been used to produce valuable rare sugars for industrial purposes. Of the Rpis, D-ribose-5-phosphate isomerase B from Clostridium thermocellum (CtRpi) has the fastest reactions kinetics.

Cloning, expression, crystallization and preliminary X-ray crystallographic analysis of the co-chaperonin XoGroES from Xanthomonas oryzae pv. oryzae.

Bacterial blight (BB), a devastating disease caused by Xanthomonas oryzae pv. oryzae (Xoo), causes serious production losses of rice in Asian countries. Protein misfolding may interfere with the function of proteins in all living cells and must be prevented to avoid cellular disaster.

Crystallization and preliminary X-ray crystallographic analysis of L-rhamnose isomerase with a novel high thermostability from Bacillus halodurans.

L-Rhamnose isomerases catalyze isomerization between L-rhamnose (6-deoxy-L-mannose) and L-rhamnulose (6-deoxy-L-fructose), which is the first step in rhamnose catabolism. L-Rhamnose isomerase from Bacillus halodurans ATCC BAA-125 (BHRI) exhibits interesting characteristics such as high thermostability and selective substrate specificity. BHRI fused with an HHHHHH sequence was purified and crystallized in order to elucidate the molecular basis of its unique enzymatic properties.

Crystallization and preliminary X-ray crystallographic analysis of DNA gyrase GyrB subunit from Xanthomonas oryzae pv. oryzae.

DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological forms of bacterial DNA. In this study, the N-terminal fragment of the GyrB subunit of DNA gyrase from Xanthomonas oryzae pv. oryzae was overexpressed in Escherichia coli, purified and crystallized.