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.

Biochemical Characterization of the Mycobacterium smegmatis Threonine Deaminase.

The biosynthesis of branched-chain amino acids or BCAAs (l-isoleucine, l-leucine, and l-valine) is essential in eubacteria, but mammals are branched-chain amino acid auxotrophs, making the enzymes in the pathway excellent targets for antibacterial drug development. The biosynthesis of l-isoleucine, l-leucine, and l-valine is very efficient, requiring only eight enzymes. Threonine dehydratase (TD), a pyridoxal 5'-phosphate (PLP)-dependent enzyme encoded by the ilvA gene, is the enzyme responsible for the conversion of l-threonine (l-Thr) to α-ketobutyrate, ammonia, and water, which is the first step in the biosynthesis of l-isoleucine. We have cloned, expressed, and biochemically characterized the reaction catalyzed by Mycobacterium smegmatis TD (abbreviated as MsIlvA) using steady-state kinetics and kinetic isotope effects. We show here that in addition to l-threonine, l-allo-threonine and l-serine are also used as substrates by TD, and all exhibit sigmoidal, non-Michaelis-Menten kinetics. Curiously, ß-chloro-l-alanine was also a substrate rather than an inhibitor as expected. The enzymatic activity of TD is sensitive to the presence of allosteric regulators, including the activator l-valine or the end product feedback inhibitor of the BCAA pathway in which TD is involved, l-isoleucine. Primary deuterium kinetic isotopes are small, suggesting Cα proton abstraction is only partially rate-limiting. Solvent kinetic isotopes were significantly larger, indicating that a proton transfer occurring during the reaction is also partially rate-limiting. Finally, we demonstrate that l-cycloserine, a general inhibitor of PLP-dependent enzymes, is an excellent inhibitor of threonine deaminase.

Increased productivity of L-2-aminobutyric acid and total turnover number of NAD+/NADH in a one-pot system through enhanced thermostability of L-threonine deaminase.

OBJECTIVE: To strengthen NADH regeneration in the biosynthesis of L-2-aminobutyric acid (L-ABA). RESULTS: L-Threonine deaminase (L-TD) from Escherichia coli K12 was modified by directed evolution and rational design to improve its endurance to heat treatment. The half-life of mutant G323D/F510L/T344A at 42 °C increased from 10 to 210 min, a 20-fold increase compared to the wild-type L-TD, and the temperature at which the activity of the enzyme decreased by 50% in 15 min increased from 39 to 53 °C. The mutant together with thermostable L-leucine dehydrogenase from Bacillus sphaericus DSM730 and formate dehydrogenase from Candida boidinii constituted a one-pot system for L-ABA biosynthesis. Employing preheat treatment in the one-pot system, the biosynthesis of L-ABA and total turnover number of NAD+/NADH were 0.993 M and 16,469, in contrast to 0.635 M and 10,531 with wild-type L-TD, respectively. CONCLUSIONS: By using the engineered L-TD during endured preheat treatment, the one-pot system has achieved a higher productivity of L-ABA and total turnover number of coenzyme.

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).

Overproduction, crystallization and preliminary X-ray crystallographic analysis of Escherichia coli tRNA N6-threonylcarbamoyladenosine dehydratase.

Escherichia coli tRNA N6-threonylcarbamoyladenosine dehydratase (TcdA), previously called CsdL or YgdL, was overproduced and purified from E. coli and crystallized using polyethylene glycol 3350 as a crystallizing agent. X-ray diffraction data were collected to 2.70 Šresolution under cryoconditions using synchrotron X-rays. The crystals belonged to space group P21, with unit-cell parameters a=65.4, b=96.8, c=83.3 Å, ß=111.7°. According to the Matthews coefficient, the asymmetric unit may contain up to four subunits of the monomeric protein, with a crystal volume per protein mass (VM) of 2.12 Å3 Da(-1) and 42.1% solvent content.

Threonine deaminase MoIlv1 is important for conidiogenesis and pathogenesis in the rice blast fungus Magnaporthe oryzae.

Threonine deaminase is the first critical enzyme in the biosynthesis of branched-chain amino acids (BCAAs), which catalyzes threonine into NH2 and ketobutyrate acid. Previously, we identified and characterized two acetolactate synthases MoIlv2 and MoIlv6 that are involved in the second step of BCAA biosynthesis. Deletion of MoILV2 and MoILV6 resulted in auxotrophy for leucine, isoleucine, and valine and defects in conidiation, appressorial penetration, and pathogenicity. Here, we identified a threonine dehydratase, named MoIlv1, from Magnaporthe oryzae. MoIlv1 is a homolog of Saccharomyces cerevisiae Ilv1p, which has an important role in the biosynthesis of isoleucine. To characterize the function of MoIlv1, a ΔMoilv1 knock-out mutant was generated and analyzed. Disruption of MoILV1 resulted in abnormal conidial morphology, reduced conidiation, limited appressorium-mediated penetration, and attenuated virulence on both barley and rice seedlings. Further analysis by domain-specific deletion revealed that the PALP domain is indispensable for MoIlv1 function. Our study indicates that MoIlv1 is a protein involved in isoleucine biosynthesis that underlies the complex process governing morphogenesis, appressorium formation, invasive hyphae growth, and pathogenicity.

The role of ACT-like subdomain in bacterial threonine dehydratases.

In bacteria, threonine dehydratases could convert L-threonine to 2-ketobutyrate. Some threonine dehydratases contain only a catalytic domain, while others contain an N-terminal catalytic domain and a C-terminal regulatory domain composed of one or two ACT-like subdomains. However, the role of the ACT-like subdomain in threonine dehydratases is not clear. Here, nine different bacterial threonine dehydratases were studied. Three of the nine contain no ACT-like subdomain, four of them contain a single ACT-like subdomain, and two of them contain two ACT-like subdomains. The nine genes encoding these threonine dehydratases were individually overexpressed in E. coli BL21(DE3), and the enzymes were purified to homogeneity. Activities of the purified enzymes were analyzed after incubation at different temperatures and different pHs. The results showed that threonine dehydratases with a single ACT-like subdomain are more stable at higher temperatures and a broad range of pH than those without ACT-like subdomain or with two ACT-like subdomains. Furthermore, the specific activity of threonine dehydratases increases with the increase of the number of ACT-like subdomains they contain. The results suggest that the ACT-like subdomain plays an important role in bacterial threonine dehydratases.

Analytical kinetic modeling: a practical procedure.

This chapter describes a practical procedure to dissect metabolic systems, simplify them, and use or derive enzyme rate equations in order to build a mathematical model of a metabolic system and run simulations. We first deal with a simple example, modeling a single enzyme that follows Michaelis-Menten kinetics and operates in the middle of an unbranched metabolic pathway. Next we describe the rules that can be followed to isolate sub-systems from their environment to simulate their behavior. Finally we use examples to show how to derive suitable rate equations, simpler than those needed for mechanistic studies, though adequate to describe the behavior over the physiological range of conditions.Many of the general characteristics of kinetic models will be obvious to readers familiar with the theory of metabolic control analysis (Cornish-Bowden, Fundamentals of Enzyme Kinetics, Wiley-Blackwell, Weinheim, 327-380, 2012), but here we shall not assume such knowledge, as the chapter is directed toward practical application rather than theory.

Molecular evolution of threonine dehydratase in bacteria.

Threonine dehydratase converts L-threonine to 2-ketobutyrate. Several threonine dehydratases exist in bacteria, but their origins and evolutionary pathway are unknown. Here we analyzed all the available threonine dehydratases in bacteria and proposed an evolutionary pathway leading to the genes encoding three different threonine dehydratases CTD, BTD1 and BTD2. The ancestral threonine dehydratase might contain only a catalytic domain, but one or two ACT-like subdomains were fused during the evolution, resulting BTD1 and BTD2, respectively. Horizontal gene transfer, gene fusion, gene duplication, and gene deletion may occur during the evolution of this enzyme. The results are important for understanding the functions of various threonine dehydratases found in bacteria.

Adaptive evolution of threonine deaminase in plant defense against insect herbivores.

Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to α-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.

Characterization of two isotypes of l-threonine dehydratase from Entamoeba histolytica.

The genome sequence of the enteric protozoan parasite Entamoeba histolytica suggests that amino acid catabolism plays an important role in energy metabolism. In the present study, we described kinetic and regulatory properties of catabolic l-threonine and l-serine dehydratase (TD) from E. histolytica. TD catalyses the pyridoxal phosphate-dependent dehydrative deamination of l-threonine and l-serine to ammonia and keto acids (2-oxobutyrate and pyruvate, respectively). E. histolytica possesses two TD isotypes (EhTD1-2) showing 38% mutual identity, a calculated molecular mass of 45.0 or 46.5kDa, and an isoelectric point of 6.68 or 5.88, respectively. Only EhTD1 showed l-threonine and l-serine dehydrative deaminating activities whereas EhTD2, in which the amino acid residues involved in the substrate and cofactor binding were not conserved, was devoid of these activities. The k(cat)/K(m) value of EhTD1 was >3 fold higher for l-threonine than l-serine. EhTD1 was inhibited by l-cysteine in a competitive manner with the K(i) values of 1.1mM and 2.2mM for l-serine and l-threonine, respectively. EhTD1 was insensitive to the allosteric activation by AMP or CMP. Three major substitutions of EhTD1 likely attribute to the insensitivity. EhTD1 was also inhibited about 50% by 20mM 2-oxobutyrate, pyruvate, and glyoxylate; the inhibition was not, however, reversed by AMP. Together, these data showed that EhTD1 possesses unique regulatory properties distinct from other organisms and may play an important role in energy metabolism via amino acid degradation in E. histolytica.

Allosteric regulation of Bacillus subtilis threonine deaminase, a biosynthetic threonine deaminase with a single regulatory domain.

The enzyme threonine deaminase (TD) is a key regulatory enzyme in the pathway for the biosynthesis of isoleucine. TD is inhibited by its end product, isoleucine, and this effect is countered by valine, the product of a competing biosynthetic pathway. Sequence and structure analyses have revealed that the protomers of many TDs have C-terminal regulatory domains, composed of two ACT-like subdomains, which bind isoleucine and valine, while others have regulatory domains of approximately half the length, composed of only a single ACT-like domain. The regulatory responses of TDs from both long and short sequence varieties appear to have many similarities, but there are significant differences. We describe here the allosteric properties of Bacillus subtilis TD ( bsTD), which belongs to the short variety of TD sequences. We also examine the effects of several mutations in the regulatory domain on the kinetics of the enzyme and its response to effectors. The behavior of bsTD can be analyzed and rationalized using a modified Monod-Wyman-Changeux model. This analysis suggests that isoleucine is a negative effector, and valine is a very weak positive effector, but that at high concentrations valine inhibits activity by competing with threonine for binding to the active site. The behavior of bsTD is contrasted with the allosteric behavior reported for TDs from Escherichia coli and Arabidopsis thaliana, TDs with two subdomains. We suggest a possible evolutionary pathway to the more complex regulatory effects of valine on the activity of TDs of the long sequence variety, e.g., E. coli TD.

Conquering isoleucine auxotrophy of Escherichia coli BLR(DE3) to recombinantly produce spider silk proteins in minimal media.

Large-scale production of recombinant spider silk proteins is a long-term goal for their industrial use. Therefore, we have recently developed a process for bacterial production. Due to a highly repetitive gene sequence of spider silks, the host strain E. coli BLR(DE3) was employed since it shows no homologue recombination. Although perfectly suited for production in full media, the BLR strain does not grow in cost-effective minimal media, indicating a previously not reported L: -isoleucine auxotrophy. We provide evidence that mutated threonine deaminase is likely responsible for the detected auxotrophy of BLR.

Crystal structures of Salmonella typhimurium biodegradative threonine deaminase and its complex with CMP provide structural insights into ligand-induced oligomerization and enzyme activation.

Two different pyridoxal 5'-phosphate-containing l-threonine deaminases (EC 4.3.1.19), biosynthetic and biodegradative, which catalyze the deamination of l-threonine to alpha-ketobutyrate, are present in Escherichia coli and Salmonella typhimurium. Biodegradative threonine deaminase (TdcB) catalyzes the first reaction in the anaerobic breakdown of l-threonine to propionate. TdcB, unlike the biosynthetic threonine deaminase, is insensitive to l-isoleucine and is activated by AMP. In the present study, TdcB from S. typhimurium was cloned and overexpressed in E. coli. In the presence of AMP or CMP, the recombinant enzyme was converted to the tetrameric form accompanied by significant enzyme activation. To provide insights into ligand-mediated oligomerization and enzyme activation, crystal structures of S. typhimurium TdcB and its complex with CMP were determined. In the native structure, TdcB is in a dimeric form, whereas in the TdcB.CMP complex, it exists in a tetrameric form with 222 symmetry and appears as a dimer of dimers. Tetrameric TdcB binds to four molecules of CMP, two at each of the dimer interfaces. Comparison of the dimer structure in the ligand (CMP)-free and -bound forms suggests that the changes induced by ligand binding at the dimer interface are essential for tetramerization. The differences observed in the tertiary and quaternary structures of TdcB in the absence and presence of CMP appear to account for enzyme activation and increased binding affinity for l-threonine. Comparison of TdcB with related pyridoxal 5'-phosphate-dependent enzymes points to structural and mechanistic similarities.

Crystallization and preliminary X-ray crystallographic analysis of biodegradative threonine deaminase (TdcB) from Salmonella typhimurium.

Biodegradative threonine deaminase (TdcB) catalyzes the deamination of L-threonine to alpha-ketobutyrate, the first reaction in the anaerobic breakdown of L-threonine to propionate. Unlike the biosynthetic threonine deaminase, TdcB is insensitive to L-isoleucine and is activated by AMP. Here, the cloning of TdcB (molecular weight 36 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli is reported. TdcB was purified to homogeneity using Ni-NTA affinity column chromatography and crystallized using the hanging-drop vapour-diffusion technique in three different crystal forms. Crystal forms I (unit-cell parameters a = 46.32, b = 55.30, c = 67.24 A, alpha = 103.09, beta = 94.70, gamma = 112.94 degrees) and II (a = 56.68, b = 76.83, c = 78.50 A, alpha = 66.12, beta = 89.16, gamma = 77.08 degrees) belong to space group P1 and contain two and four molecules of TdcB, respectively, in the asymmetric unit. Poorly diffracting form III crystals were obtained in space group C2 and based on the unit-cell volume are most likely to contain one molecule per asymmetric unit. Two complete data sets of resolutions 2.2 A (crystal form I) and 1.7 A (crystal form II) were collected at 100 K using an in-house X-ray source.

A site-directed mutagenesis interrogation of the carboxy-terminal end of Arabidopsis thaliana threonine dehydratase/deaminase reveals a synergistic interaction between two effector-binding sites and contributes to the development of a novel selectable marker.

We fused four mutant omr1 alleles, encoding feedback-insensitive forms of Arabidopsis thaliana biosynthetic threonine dehydratase/deaminase (TD), to the CaMV 35S promoter and transformed these constructs into A. thaliana Columbia wild type plants. The mutant TD forms consisted of our previously isolated double mutant, omr1-1 , and three new site-directed mutants, omr1-5 , omr1-7 , and omr1-8 with single point mutations. We employed site-directed mutagenesis to assay the effects of amino acid substitutions in separate regulatory regions within the carboxy-terminal (C-term) allosteric end. TD assays and growth resistance to the isoleucine (Ile) toxic analog -O-methylthreonine (OMT) confirmed the desensitization to feedback inhibition and the viability of these mutant omr1 alleles as selectable markers, respectively. Two of the site-directed mutants, omr1-5 and omr1-7 , appeared to influence one of the two separate Ile-binding sites and had a notable 13-fold and 15-fold increase in free Ile, respectively. The omr1-8 appeared to influence the other Ile-binding site and resulted in a 2-fold increase in free Ile. The transgenic omr1-1 double mutant affecting both Ile-binding sites, however, displayed a 106-fold increase in free Ile revealing a profound synergistic interplay between these separate Ile-binding sites. While all of the four omr1 alleles conferred resistance to elevated concentrations of OMT, the progeny of omr1-1 initial transformants exhibited a bushy phenotype at the rosette stage. On the other hand, progeny of transformants omr1-5 , omr1-7 , and omr1-8 had a normal phenotype, undistinguishable from wild type. Therefore, alleles omr1-5 , omr1-7 , and omr1-8 , proved to be ideal as environmentally-friendly, dominant, selectable markers for plant transformation.

Structure and function correlations between the rat liver threonine deaminase and aminotransferases.

The rat liver threonine deaminase is a cytoplasmic enzyme that catalyses the pyridoxal-phosphate-dependent dehydrative deamination of L-threonine and L-serine to ammonia and alpha-ketobutyrate and pyruvate, respectively, in vivo. During deamination, a molecule of the cofactor is converted to pyridoxamine phosphate. Recently, the ability of this enzyme to accomplish an inverse half-reaction, restoring pyridoxal-phosphate and L-alanine or L-aminobutyrate, respectively, from pyruvate or 2-oxobutyrate, was reported. In order to investigate the molecular mechanisms of this transaminating activity, a molecular model of rat liver threonine deaminase was constructed on the basis of sequence homology with the biosynthetic threonine deaminase of Escherichia coli, the crystal structure of which is known. The model has structural features shared by aminotransferases, suggesting that tertiary structural elements may be responsible for the transaminating activity observed for rat liver threonine deaminase.

Biochemical and mass spectrometric evidence for quaternary structure modifications of plant threonine deaminase induced by isoleucine.

Arabidopsis thaliana threonine deaminase (TD) is a tetramer composed of identical approximately 59600 Da subunits. TD activity has been shown to be inhibited by isoleucine. This effect is reversed by a large excess of valine. Nondenaturant gel filtration, polyacrylamide gel electrophoresis, and mass spectrometry experiments demonstrated that binding of isoleucine on TD induces dimerization of the enzyme, whereas tetramerization is restored by addition of a high valine concentration. Nondenaturant gel filtration and electrospray ionization mass spectrometry of the enzyme in the presence of increasing amounts of isoleucine suggest a fast equilibrium between the tetramer and the dimer. Finally, study of TD mutants allowed us to focus on the specific role of each isoleucine-binding site.

The ACT domain family.

A novel ligand-binding domain, named the 'ACT domain', was recently identified by a PSI-BLAST search. The archetypical ACT domain is the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase (3PGDH), which folds with a ferredoxin-like betaalphabetabetaalphabeta topology. A pair of ACT domains form an eight-stranded antiparallel sheet with two molecules of the allosteric inhibitor serine bound in the interface. The ACT domain is found in a variety of contexts and is proposed to be a conserved regulatory ligand binding fold. Rat phenylalanine hydroxylase has a regulatory domain with a similar fold, but different ligand-binding mode. Putative ACT domains in some proteins of unknown structure (e.g. acetohydroxyacid synthase regulatory subunits) may also fold like the 3PGDH regulatory domain. The regulatory domain of threonine deaminase, although not a member of the ACT sequence family, is similar in structure to the paired 3PGDH regulatory domains. Repeats of ACT-like domains can create nonequivalent ligand-binding sites with the potential for complex regulatory patterns. The structures and mechanisms of such systems have only begun to be examined.

New approach to analysis of deviations from hyperbolic law in enzyme kinetics.

An empirical equation that describes deviations from Michaelian kinetics is proposed. The equation allows the limiting values of the Michaelis constant at v/Vmax --> 0 and v/Vmax --> 1 to be estimated (v is the rate of the enzymatic reaction and Vmax is the limiting value of v at saturating concentrations of substrate). The applicability of the equation is demonstrated for kinetic data obtained for glutamate dehydrogenases from various sources (negative kinetic cooperativity for coenzyme) and for biosynthetic threonine deaminase from pea seedlings (sharper approaching the limiting value of the enzymatic reaction rate with increasing substrate concentration in comparison with the hyperbolic law). The negative cooperativity for the function of saturation of protein by ligand is also analyzed (data on binding of spin-labeled NAD, NADH, and NADPH by beef liver glutamate dehydrogenase and binding of cupric ions by BSA are used as examples).