Identification and biochemical characterization of threonine dehydratase from the hyperthermophile Thermotoga maritima.

The peptidoglycan of the hyperthermophile Thermotoga maritima contains an unusual component, D-lysine (D-Lys), in addition to the typical D-alanine (D-Ala) and D-glutamate (D-Glu). In a previous study, we identified a Lys racemase that is presumably associated with D-Lys biosynthesis. However, our understanding of D-amino acid metabolism in T. maritima and other bacteria remains limited, although D-amino acids in the peptidoglycan are crucial for preserving bacterial cell structure and resistance to environmental threats. Herein, we characterized enzymatic and structural properties of TM0356 that shares a high amino acid sequence identity with serine (Ser) racemase. The results revealed that TM0356 forms a tetramer with each subunit containing a pyridoxal 5'-phosphate as a cofactor. The enzyme did not exhibit racemase activity toward various amino acids including Ser, and dehydratase activity was highest toward L-threonine (L-Thr). It also acted on L-Ser and L-allo-Thr, but not on the corresponding D-amino acids. The catalytic mechanism did not follow typical Michaelis-Menten kinetics; it displayed a sigmoidal dependence on substrate concentration, with highest catalytic efficiency (kcat/K0.5) toward L-Thr. Interestingly, dehydratase activity was insensitive to allosteric regulators L-valine and L-isoleucine (L-Ile) at low concentrations, while these L-amino acids are inhibitors at high concentrations. Thus, TM0356 is a biosynthetic Thr dehydratase responsible for the conversion of L-Thr to α-ketobutyrate and ammonia, which is presumably involved in the first step of the biosynthesis of L-Ile.

Threonine dehydratase enhances bacterial cadmium resistance via driving cysteine desulfuration and biomineralization of cadmium sulfide nanocrystals.

Biomineralization is often used by microorganisms to sequester heavy metal ions and provides a potential means for remediating increasing levels of heavy metal pollution. Bacteria have been shown to utilize cysteine for the biomineralization of metal sulfide. Indeed, in the present study, the supplement of L-cysteine was found to significantly improve both cadmium resistance and removal abilities of a deep-sea bacterium Pseudomonas stutzeri 273 through cadmium sulfide (CdS) nanoparticle biomineralization. With a proteomic approach, threonine dehydratase of P. stutzeri 273 (psTD) was proposed to be a key factor enhancing bacterial cadmium resistance through catalyzing L-cysteine desulfuration, H2S generation and CdS nanoparticle biomineralization. Consistently, deletion of the gene encoding psTD in P. stutzeri 273 resulted in the decline of H2S generation, decrease of cadmium resistance, and reduction of cadmium removal ability, confirming the unique function of psTD directing the formation of CdS nanoparticles. Correspondingly, the single-enzyme biomineralization of CdS nanoparticle driven by psTD was further developed, and psTD was shown to act as a capping reagent for the mineralization reaction, which controlling the size and structure of nanocrystals. Our results provide important clues for the construction of engineered bacteria for cadmium bioremediation and widen the synthesis methods of nanomaterials.

Analyses of variants of the Ser/Thr dehydratase IlvA provide insight into 2-aminoacrylate metabolism in Salmonella enterica.

RidA is a conserved and broadly distributed protein that has enamine deaminase activity. In a variety of organisms tested thus far, lack of RidA results in the accumulation of the reactive metabolite 2-aminoacrylate (2AA), an obligate intermediate in the catalytic mechanism of several pyridoxal 5'-phosphate (PLP)-dependent enzymes. This study reports the characterization of variants of the biosynthetic serine/threonine dehydratase (EC 4.3.1.19; IlvA), which is a significant generator of 2AA in the bacteria Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa and the yeast Saccharomyces cerevisiae Two previously identified mutations, ilvA3210 and ilvA3211, suppressed the phenotypic growth consequences of 2AA accumulation in S. enterica Characterization of the respective protein variants suggested that they affect 2AA metabolism in vivo by two different catalytic mechanisms, both leading to an overall reduction in serine dehydratase activity. To emphasize the physiological relevance of the in vitro enzyme characterization, we sought to explain in vivo phenotypes using these data. A simple mathematical model describing the impact these catalytic deficiencies had on 2AA production was generally supported by our data. However, caveats arose when kinetic parameters, determined in vitro, were used to predict formation of the isoleucine precursor 2-ketobutyrate and model in vivo (growth) behaviors. Altogether, our data support the need for a holistic approach, including in vivo and in vitro analyses, to generate data used in understanding and modeling metabolism.

Involvement of C-terminal amino acids of a hyperthermophilic serine racemase in its thermostability.

Pyrobaculum islandicum is a hyperthermophilic archaeon that grows optimally at 95-100 °C. In the previous study, we extensively purified a serine racemase from this organism and cloned the gene for overexpression in Escherichia coli (Ohnishi et al. 2008). This enzyme also exhibits highly thermostable L-serine/L-threonine dehydratase activity. In the present study, we aimed to elucidate the molecular mechanisms underlying the high thermostability of this enzyme. A recombinant variant of this enzyme, PiSRvt, constructed by truncating the C-terminal 72 amino acids, was compared with the native enzyme, PiSR. The dehydratase activity of PiSR and PiSRvt was found to owe to a homotrimer and a monomer, respectively, that demonstrated high and moderate thermostability, respectively. These observations reveal that the C-terminal region contributes to monomer trimerization that provides the extreme thermostability.

Biochemical and functional characterization of MRA_1571 of Mycobacterium tuberculosis H37Ra and effect of its down-regulation on survival in macrophages.

Amino acid biosynthesis has emerged as a source of new drug targets as many bacterial strains auxotrophic for amino acids fail to proliferate under in vivo conditions. Branch chain amino acids (BCAAs) are important for Mycobacterium tuberculosis (Mtb) survival and strains deficient in their biosynthesis were attenuated for growth in mice. Threonine dehydratase (IlvA) is a pyridoxal-5-phosphate (PLP) dependent enzyme that catalyzes the first step in isoleucine biosynthesis. The MRA_1571 of Mycobacterium tuberculosis H37Ra (Mtb-Ra), annotated to be coding for IlvA, was cloned, expressed and purified. Purified protein was subsequently used for developing enzyme assay and to study its biochemical properties. Also, E. coli BL21 (DE3) IlvA knockout (E. coli-ΔilvA) was developed and genetically complemented with Mtb-Ra ilvA expression construct (pET32a-ilvA) to make complemented E. coli strain (E. coli-ΔilvA + pET32a-ilvA). The E. coli-ΔilvA showed growth failure in minimal medium but growth restoration was observed in E. coli-ΔilvA + pET32a-ilvA. E. coli-ΔilvA growth was also restored in the presence of isoleucine. The IlvA localization studies detected its distribution in cell wall and membrane fractions with relatively minor presence in cytosolic fraction. Maximum IlvA expression was observed at 72 h in wild-type (WT) Mtb-Ra infecting macrophages. Also, Mtb-Ra IlvA knockdown (KD) showed reduced survival in macrophages compared to WT and complemented strain (KDC).

Production of α-ketobutyrate using engineered Escherichia coli via temperature shift.

Alpha-ketobutyrate has been widely used in medicine and food additive industry. Because chemical and enzymatic methods are associated with many deficiencies, the recent focus shifted to fermentation for the production of α-ketobutyrate. In this study, a genetically engineered strain THRDΔrhtAΔilvIH/pWSK29-ilvA was constructed, starting from an L-threonine-producing strain, by overexpressing threonine dehydratase (TD), reducing α-ketobutyrate catabolism and L-threonine export. The shake flask cultivation of THRDΔrhtAΔilvIH/pWSK29-ilvA allowed the production of 16.2 g/L α-ketobutyrate. Accumulation of α-ketobutyrate severely inhibited the cell growth. To develop a better TD expression system and avoid the usage of the expensive inducer IPTG, a temperature-induced plasmid pBV220-ilvA was selected to transform the strain THRDΔrhtAΔilvIH for α-ketobutyrate production. The initial temperature was maintained at 35°C to guarantee normal cell growth, and then elevated to 40°C to induce the expression of TD. Under optimized conditions, the α-ketobutyrate titer reached 40.8 g/L after 28 h of fermentation, with a productivity of 1.46 g/L/h and a yield of 0.19 g/g glucose, suggesting large-scale production potential. Biotechnol. Bioeng. 2016;113: 2054-2059. © 2016 Wiley Periodicals, Inc.

Responses in whole-body amino acid kinetics to an acute, sub-clinical endotoxin challenge in lambs.

Some effects of parasitism, endotoxaemia or sepsis can be mitigated by provision of extra protein. Supplemented protein may encompass a metabolic requirement for specific amino acids (AA). The current study investigates a method to identify and quantify the amounts of AA required during inflammation induced by an endotoxin challenge. One of each pair of six twin sheep was infused in the jugular vein for 20 h with either saline (control) or lipopolysaccharide (LPS, 2 ng/kg body weight per min) from Escherichia coli. Between 12 and 20 h a mixture of stable isotope-labelled AA was infused to measure irreversible loss rates. From 16 to 20 h all sheep were supplemented with a mixture of unlabelled AA infused intravenously. Blood samples were taken before the start of infusions, and then continuously over intervals between 14 and 20 h. At 20 h the sheep were euthanised, and liver and kidney samples were taken for measurement of serine-threonine dehydratase (SDH) activity. LPS infusion decreased plasma concentrations of most AA (P<0·05; P<0·10 for leucine and tryptophan), except for phenylalanine (which increased P=0·022) and tyrosine. On the basis of the incremental response to the supplemental AA, arginine, aspartate, cysteine, glutamate, lysine (tendency only), glycine, methionine, proline, serine and threonine were important in the metabolic response to the endotoxaemia. The AA infusion between 16 and 20 h restored the plasma concentrations in the LPS-treated sheep for the majority of AA, except for glutamine, isoleucine, methionine, serine and valine. LPS treatment increased (P<0·02) SDH activity in both liver and kidney. The approach allows quantification of key AA required during challenge situations.