Mechanism of action of D-serine dehydratase. Identification of a transient intermediate.

Static absorbance measurements of D-serine dehydratase from Escherichia coli taken at 2 degrees C show that during the steady-state course of D-serine conversion the absorption maximum of the Schiff base of the cofactor pyridoxal 5'-phosphate (pyridoxal-P) is shifted from 415 to 442 nm. Furthermore, the progress curve of intermediates was monitored by stopped-flow techniques at wavelengths ranging from 320 to 500 nm. A point by point construction of successive spectra from these stopped-flow traces at various time intervals after the start of reaction resulted in a series of consecutive spectra exhibiting two isobestic points at 353 and 419 nm. The half-time of the absorbance changes occurring at 330 and 455 nm was found to be 6.5 ms, suggesting the observation of a single, enzyme-bound intermediate. The spectral data with substrate and inhibitors provide evidence that the intermediate is the Schiff base of alpha-aminoacrylate and pyridoxal-P. The proposed assignment is strongly supported by experiments of apodehydratase with transient-state analogues which exhibit a similar absorbance shift on binding to apoenzyme. Moreover, these results suggest that the phosphate group of the substrate--pyridoxal-P complex serves as the main anchoring point during catalysis. A reaction mechanism of the D-serine dehydratase is presented.     

Construction of carotenoid biosynthetic pathways using squalene synthase

The first committed steps of steroid/hopanoid pathways involve squalene synthase (SQS). Here, we report the Escherichia coli production of diaponeurosporene and diapolycopene, yellow C₃₀ carotenoid pigments, by expressing human SQS and Staphylococcus aureus dehydrosqualene (C₃₀ carotenoid) desaturase (CrtN). We suggest that the carotenoid pigments are synthesized mainly via the desaturation of squalene rather than the direct synthesis of dehydrosqualene through the non-reductive condensation of prenyl diphosphate precursors, indicating the possible existence of a “squalene route” and a “lycopersene route” for C₃₀ and C₄₀ carotenoids, respectively. Additionally, this finding yields a new method of colorimetric screening for the cellular activity of squalene synthases, which are major targets for cholesterol-lowering drugs.     

Crystal structure of a zinc-dependent D-serine dehydratase from chicken kidney

d-Serine is a physiological co-agonist of the N-methyl-d-aspartate receptor. It regulates excitatory neurotransmission, which is important for higher brain functions in vertebrates. In mammalian brains, d-amino acid oxidase degrades d-serine. However, we have found recently that in chicken brains the oxidase is not expressed and instead a d-serine dehydratase degrades d-serine. The primary structure of the enzyme shows significant similarities to those of metal-activated d-threonine aldolases, which are fold-type III pyridoxal 5′-phosphate (PLP)-dependent enzymes, suggesting that it is a novel class of d-serine dehydratase. In the present study, we characterized the chicken enzyme biochemically and also by x-ray crystallography. The enzyme activity on d-serine decreased 20-fold by EDTA treatment and recovered nearly completely by the addition of Zn²⁺. None of the reaction products that would be expected from side reactions of the PLP-d-serine Schiff base were detected during the >6000 catalytic cycles of dehydration, indicating high reaction specificity. We have determined the first crystal structure of the d-serine dehydratase at 1.9 Å resolution. In the active site pocket, a zinc ion that coordinates His³⁴⁷ and Cys³⁴⁹ is located near the PLP-Lys⁴⁵ Schiff base. A theoretical model of the enzyme-d-serine complex suggested that the hydroxyl group of d-serine directly coordinates the zinc ion, and that the ϵ-NH₂ group of Lys⁴⁵ is a short distance from the substrate Cα atom. The α-proton abstraction from d-serine by Lys⁴⁵ and the elimination of the hydroxyl group seem to occur with the assistance of the zinc ion, resulting in the strict reaction specificity.     

Co-expression of feedback-resistant threonine dehydratase and acetohydroxy acid synthase increase l-isoleucine production in Corynebacterium glutamicum

Threonine dehydratase and acetohydroxy acid synthase are critical enzymes in the l-isoleucine biosynthesis pathway of Corynebacterium glutamicum, but their activities are usually feedback-inhibited. In this study, we characterized a feedback-resistant threonine dehydratase and an acetohydroxy acid synthase from an l-isoleucine producing strain C. glutamicum JHI3-156. Sequence analysis showed that there was only a single amino acid substitution (Phe383Val) in the feedback-resistant threonine dehydratase, and there were three mutated amino acids (Pro176Ser, Asp426Glu, and Leu575Trp) in the big subunit of feedback-resistant acetohydroxy acid synthase. The mutated threonine dehydratase over-expressed in E. coli not only showed completely resistance to l-isoleucine inhibition, but also showed enhanced activity. The mutated acetohydroxy acid synthase over-expressed in E. coli showed more resistance to l-isoleucine inhibition than the wild type. Over-expression of the feedback-resistant threonine dehydratase or acetohydroxy acid synthase in C. glutamicum JHI3-156 led to increase of l-isoleucine production; co-expression of them in C. glutamicum JHI3-156 led to 131.7% increase in flask cultivation, and could produce 30.7g/L l-isoleucine in 72-h fed-batch fermentation. These results would be useful to enhance l-isoleucine production in C. glutamicum.     

The evolution of the histidine biosynthetic genes in prokaryotes: A common ancestor for the hisA and hisF genes

The hisA and hisF genes belong to the histidine operon that has been extensively studied in the enterobacteria Escherichia coli and Salmonella typhimurium where the hisA gene codes for the phosphoribosyl-5-amino-1-phosphoribosyl-4-imidazolecarboxamide isomerase (EC 5.3.1.16) catalyzing the fourth step of the histidine biosynthetic pathway, and the hisF gene codes for a cyclase catalyzing the sixth reaction. Comparative analysis of nucleotide and predicted amino acid sequence of hisA and hisF genes in different microorganisms showed extensive sequence homology (43% considering similar amino acids), suggesting that the two genes arose from an ancestral gene by duplication and subsequent evolutionary divergence. A more detailed analysis, including mutual information, revealed an internal duplication both in hisA and hisF genes in each of the considered microorganisms. We propose that the hisA and hisF have originated from the duplication of a smaller ancestral gene corresponding to half the size of the actual genes followed by rapid evolutionary divergence. The involvement of gene elongation, gene duplication, and gene fusion in the evolution of the histidine biosynthetic genes is also discussed. Correspondence to: M. Bazzicalupo     

Requirements for induction of the biodegradative threonine dehydratase in Escherichia coli.

Synthesis of the biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, was prevented by dissolved oxygen concentrations of 6 micrometer or greater. This effect was shown to be exerted solely on synthesis, rather than being the result of enzyme inactivation in vivo. In addition to an anaerobic environment, maximum enzyme synthesis was dependent upon the presence of a complete complement of amino acids, with omission of L-threonine, L-valine, or L-leucine producing the largest decreases in enzyme formation. L-Threonine, the most essential of the amino acid requirements, could be partially replaced by DL-allothreonine or alpha-ketobutyrate. Half-maximal stimulation of enzyme synthesis occurred with 0.4 mM threonine in the medium. The roles of anaerobiosis and amino acids are interpreted as being in accord with the concept that threonine dehydratase functions in anaerobic energy production under conditions of amino acid sufficiency.     

SP0454, a putative threonine dehydratase, is required for pneumococcal virulence in mice

Increasing pressure in antibiotic resistance and the requirement for the design of new vaccines are the objectives of clarifying the putative virulence factors in pneumococcal infection. In this study, the putative threonine dehydratase sp0454 was inactivated by erythromycin-resistance cassette replacement in Streptococcus pneumoniae CMCC 31203 strain. The sp0454 mutant was tested for cell growth, adherence, colonization, and virulence in a murine model. The Δsp0454 mutant showed decreased ability for colonization and impaired ability to adhere to A549 cells. However, the SP0454 polypeptide or its antiserum did not affect pneumococcal CMCC 31203 adhesion to A549 cells. The sp0454 deletion mutant was less virulent in a murine intranasal infection model. Real-time RT-PCR analysis revealed significant decrease of the pneumococcal surface antigen A expression in the sp0454 mutant. These results suggest that SP0454 contributes to virulence and colonization, which could be explained in part by modulating the expression of other virulence factors, such as psaA in pneumococcal infection.     

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.     

Plant lanosterol synthase: divergence of the sterol and triterpene biosynthetic pathways in eukaryotes

Sterols, essential eukaryotic constituents, are biosynthesized through either cyclic triterpenes, lanosterol (fungi and animals) or cycloartenol (plants). The cDNA for OSC7 of Lotus japonicus was shown to encode lanosterol synthase (LAS) by the complementation of a LAS-deficient mutant yeast and structural identification of the accumulated lanosterol. A double site-directed mutant of OSC7, in which amino acid residues crucial for the reaction specificity were changed to the cycloartenol synthase (CAS) type, produced parkeol and cycloartenol. The multiple amino acid sequence alignment of a conserved region suggests that the LAS of different eukaryotic lineages emerged from the ancestral CAS by convergent evolution.     

Functional and structural analyses of threonine dehydratase from Corynebacterium glutamicum.

Threonine dehydratase activity is an important element in the flux control of isoleucine biosynthesis. The enzyme of Corynebacterium glutamicum demonstrates a marked sigmoidal dependence of initial velocity on the threonine concentration, a dependence that is consistent with substrate-promoted conversion of the enzyme from a low-activity to a high-activity conformation. In the presence of the negative allosteric effector isoleucine, the K0.5 increased from 21 to 78 mM and the cooperativity, as expressed by the Hill coefficient increased from 2.4 to 3.7. Valine promoted opposite effects: the K0.5 was reduced to 12 mM, and the enzyme exhibited almost no cooperativity. Sequence determination of the C. glutamicum gene for this enzyme revealed an open reading frame coding for a polypeptide of 436 amino acids. From this information and the molecular weight determination of the native enzyme, it follows that the dehydratase is a tetramer with a total mass of 186,396 daltons. Comparison of the deduced polypeptide sequence with the sequences of known threonine dehydratases revealed surprising differences from the C. glutamicum enzyme in the carboxy-terminal portion. This portion is greatly reduced in size, and a large gap of 95 amino acids must be introduced to achieve homology. Therefore, the C. glutamicum enzyme must be considered a small variant of threonine dehydratase that is typically controlled by isoleucine and valine but has an altered structure reflecting a topological difference in the portion of the protein most likely to be important for allosteric regulation.     

Catabolite inactivation of biodegradative threonine dehydratase of Escherichia coli.

Incubation of Escherichia coli cells with glucose, pyruvate, and certain other metabolites led to rapid inactivation of inducible biodegradative threonine dehydratase. Analysis with several mutant strains showed that pyruvate, and not a metabolite derived from pyruvate, was capable of inactivating enzyme, and that glucose acted indirectly after being converted to pyruvate. Some other alpha-keto acids such as oxaloacetate and alpha-ketobutyrate (but not alpha-ketoglutarate) were also effective. Inactivation of threonine dehydratase by pyruvate was also observed with purified enzyme preparations. The rates of enzyme inactivation increased with increased concentrations of pyruvate and decreased with increased levels of AMP. Increasing protein concentrations lowered the rates of enzyme inactivation. Dithiothreitol had a large effect on the maximum extent of inactivation of the enzyme by pyruvate; high concentrations of AMP and DTT almost completely counteracted the effect of pyruvate. Gel filtration data showed that pyruvate influenced the oligomeric state of the enzyme by altering the association-dissociation equilibrium in favor of dissociation; the Stokes' radius of the pyruvate-inactivated enzyme was 32 A as compared to 42 A for the untreated enzyme. Reassociation of the dissociated form of the enzyme was achieved by removal of excess free pyruvate by dialysis against buffer supplemented with AMP and DTT. Incubation of threonine dehydratase with [14-C]pyruvate revealed apparent covalent attachment of pyruvate to the enzyme. Strong protein denaturants such as guanidine, urea, and sodium dodecyl sulfate failed to release bound radioactive pyruvate; the molar ratio of firmly bound pyruvate was approximately 1 mol/150,000 g of protein. Pretreatment of the enzyme with p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) did not reduce the binding of [14-C]pyruvate suggesting no active site SH was involved in the pyruvate-enzyme linkage. Titration of active and pyruvate-inactivated enzyme with Nbs2 indicated that the loss in enzyme activity was not due to oxidation of essential sulfhydryl groups on the enzyme. Based on these data we propose that the mechanism of enzyme inactivation by pyruvate involves covalent attachment of pyruvate to the active oligomeric form of the enzyme followed by dissociation of the oligomer to yield inactive enzyme.     

13C NMR spectroscopy of labeled pyridoxal 5'-phosphate. Model studies, D-serine dehydratase, and L-glutamate decarboxylase.

Pyridoxal 5'-phosphate labeled to the extent of 90% with 13C in the 4' (aldehyde) and 5' (methylene) positions has been synthesized. 13C NMR spectra of this material and of natural abundance pyridoxal 5'-phosphate are reported, as well as 13C NMR spectra of the Schiff base formed by reaction of pyridoxal 5'-phosphate with n-butylamine, the secondary amine formed by reduction of this Schiff base, the thiazolidine formed by reaction of pyridoxal 5'-phosphate with cysteine, the hexahydropyrimidine formed by reaction of pyridoxal 5'-phosphate with 1,3-diaminobutane, and pyridoxamine 5'-phosphate. The range of chemical shifts for carbon 4' in these compounds is more than 100 ppm, and thus this chemical shift is expected to be a sensitive indicator of structure in enzyme-bound pyridoxal 5'-phosphate. The chemical shift of carbon 5', on the other hand, is insensitive to these structure changes. 13C NMR spectra have been obtained at pH 7.8 and 9.4 for D-serine dehydratase (Mr = 46,000) containing natural abundance pyridoxal 5'-phosphate and containing 13C-enriched pyridoxal 5'-phosphate. The enriched material contains two new resonances not present in the natural abundance material, one at 167.7 ppm with a linewidth of approximately 24 Hz, attributed to carbon 4' of the Schiff base in the bound coenzyme, and one at 62.7 Hz with a linewidth of approximately 48 Hz attributed to carbon 5' of the bound Schiff base. A large number of resonances due to individual amino acids are assigned. The NMR spectrum changes only slightly when the pH is raised to 9.4. The widths of the two enriched coenzyme resonances indicate that the coenzyme is rather rigidly bound to the enzyme but probably has limited motional freedom relative to the protein. 13C NMR spectra have been obtained for L-glutamate decarboxylase containing natural abundance pyridoxal 5'-phosphate and 13C-enriched pyridoxal 5'-phosphate. Under conditions where the two enriched 13C resonances are clearly visible in D-serine dehydratase, no resonances are visible in enriched L-glutamate decarboxylase, presumably because the coenzyme is rigidly bound to the protein and the 300,000 molecular weight of this enzyme produces very short relaxation times for the bound coenzyme and thus very broad lines.     

Phosphorus-31 nuclear magnetic resonance study of D-serine dehydratase: pryridoxal phosphate binding site.

The pyridoxal phosphate dependent enzyme D-serine dehydratase has been investigated using 31P nuclear magnetic resonance (NMR) at 72.86 MHz. In the native enzyme, the pyridoxal phosphate 31P chemical shift is pH dependent with pKa = 6.4, indicating exposure of the phosphate group to solvent. Binding of the competitive inhibitor isoserine results in the formation of the isoserine-pyridoxal phosphate complex. This transaldimination complex is fixed to the enzyme via the phosphate group of the cofactor as the dianion, independent of pH. At pH 6.6 the dissociation constant KD for isoserine determined by NMR is 0.43 mM. Reconstitution of the apoenzyme with pyridoxal phosphate monomethyl ester produces an inactive enzyme. NMR and fluorescence measurements show that this enzyme does not form the transaldimination complex, indicating that the fixation of the dianionic phosphate (probably via a salt bridge with an arginine residue) observed in the native enzyme is required for the transaldimination step of the catalytic mechanism.     

Search for the wild ancestor of buckwheat III. The wild ancestor of cultivated common buckwheat, and of tatary buckwheat

By surveying wild Fagopyrum species and their distribution in southern China and the Himalayan hills, I arrived at the conclusion that the newly discovered subspecies F. esculentum ssp. ancestralis Ohnishi is the wild ancestor of cultivated common buckwheat, while previously known wild tatary buckwheat,F. tataricum ssp. potanini Batalin is the wild ancestor of tatary buckwheat. Their original birthplace is revealed to be northwestern corner of Yunnan province for common buckwheat judging from the distribution of wild ancestor, and to be the northwest part of Sichuan province for tatary buckwheat judging from allozyme variability in wild tatary buckwheat. F. cymosum is not the ancestor of cultivated buckwheat; it is only distantly related to cultivated buckwheat, in morphology, isozymes and cpDNA. Several genetic, ecological and taxonomic categories which should be taken into consideration in examining the origin of buckwheat were discussed. Key Words: Fagopyrum esculentum ssp. ancestralis; Fagopyrum tataricum ssp. potanini; southern China theory of origin of buckwheat. Contribution from Plant Germ-Plasm Institute, Faculty of Agriculture, Kyoto University. No. 78.     

Crystallization and preliminary X-ray studies of D-serine dehydratase from Escherichia coli

Single crystals of D-serine dehydratase from Escherichia coli complexed with 3-amino-2-hydroxypropionate have been obtained from ammonium sulfate solution (pH 7.0) by vapor diffusion. The crystals belong to the trigonal space group P3(1) or P3(2) with a = b = 81.3 A and c = 58.4 A. The asymmetric unit cell contains one protein molecule with Mr = 48,289. The crystals diffract to at least 3.0 A resolution and are suitable for X-ray structure analysis.     

Altered expression of biodegradative threonine dehydratase in Escherichia coli mutants.

A number of strains of Escherichia coli K-12 failed to synthesize significant amounts of biodegradative threonine dehydratase (EC 4.2.1.16) when grown anaerobically in tryptone-yeast extract medium, a condition which is optimal for the induction of this enzyme. However, the addition of 10 mM potassium nitrate to the culture medium enabled a few of these strains, notably MB201, to induce the enzyme. An examination of the kinetic parameters, modifier sensitivity, and immunological cross-reactivity revealed that the enzyme produced by MB201 in nitrate-supplemented medium appeared indistinguishable from the dehydratase of a wild-type strain. The reduced expression of threonine dehydratase in MB201 appeared highly specific; the synthesis of two other inducible enzymes, D-serine deaminase and tryptophanase, and two "anaerobic" proteins, namely, fumarate reductase and cytochrome c551, remained unaffected. The mutation (tdcI) responsible for the altered expression of the dehydratase in MB201 was located at min 91 on the E. coli chromosome and appeared to tightly linked to if not identical with pgi, the gene encoding phosphoglucose isomerase, as judged by growth experiments on glucose and fructose, direct assay of phosphoglucose isomerase activity, spontaneous and simultaneous reversion of MB201 (tdcI) to TdcI+ and Pgi+ phenotype, and cosegregation of the two loci during transduction with P1 phage. Because not all strains lacking the dehydratase showed nitrate-dependent enzyme synthesis or had lesions at the pgi locus, it appears that mutations at multiple loci on the E. coli chromosome may influence the expression of the enzyme in vivo.     

Relationship between threonine dehydratase and biosynthesis of tylosin in Streptomyces fradiae.

To elucidate the repression mechanism of ammonium ions on the biosynthesis of tylosin in Streptomyces fradiae NRRL 2702, enzyme activities involved in the metabolism of the aspartate family of amino acids were evaluated in relation to the ammonium ion concentration and tylosin production. It was found that aspartate aminotransferase was essential for both cell growth and tylosin production. However, both threonine dehydratase and valine dehydrogenase were repressed by supplemented ammonium ions at concentrations higher than 50 mM. Threonine dehydratase was purified from cell-free extracts by acetone precipitation, ion-exchange chromatography and gel filtration, and its molecular mass was estimated to be 67,200 Da. The optimum pH and temperature for threonine dehydratase activity were 7.5 and 25 degrees C, respectively, and the Km value for threonine under these optimum conditions was 21 mM. The inhibition pattern of ammonium ions on the activity of threonine dehydratase appeared to be a mixed type.