Rat liver serine dehydratase. Bacterial expression and two folding domains as revealed by limited proteolysis.
A pCW vector harboring rat liver serine dehydratase cDNA was expressed in Escherichia coli. The expressed level was about 5-fold higher in E. coli BL21 than in JM109 cell extract; the former lacked two kinds of proteases. Immunoblot analysis revealed the occurrence of a derivative other than serine dehydratase in the JM109 cell extract. The recombinant enzyme was purified to homogeneity. Staphylococcus aureus V8 protease and trypsin cleaved the enzyme at Glu-206 and Lys-220, respectively, with a concomitant loss of enzyme activity. Spectrophotometrically, the nicked enzyme showed a approximately 50% reduced capacity for binding of the coenzyme pyridoxal phosphate and no spectral change of circular dichroism in the region at 300-480 nm, whereas circular dichroism spectra of both enzymes in the far-UV region were similar, suggesting that proteolysis impairs the coenzyme binding without an accompanying gross change of the secondary structure. Whereas the nicked enzyme behaved like the intact enzyme on Sephadex G-75 column chromatography, it was dissociated into two fragments on the column containing 6 M urea. Upon the removal of urea, both fragments spontaneously refolded. These results suggest that serine dehydratase consists of two folding domains connected by a region that is very susceptible to proteases. (+info)
Flux of the L-serine metabolism in rat liver. The predominant contribution of serine dehydratase.
L-Serine metabolism in rat liver was investigated, focusing on the relative contributions of the three pathways, one initiated by L-serine dehydratase (SDH), another by serine:pyruvate/alanine:glyoxylate aminotransferase (SPT/AGT), and the other involving serine hydroxymethyltransferase and the mitochondrial glycine cleavage enzyme system (GCS). Because serine hydroxymethyltransferase is responsible for the interconversion between serine and glycine, SDH, SPT/AGT, and GCS were considered to be the metabolic exits of the serine-glycine pool. In vitro, flux through SDH was predominant in both 24-h starved and glucagon-treated rats. Flux through SPT/AGT was enhanced by glucagon administration, but even after the induction, its contribution under quasi-physiological conditions (1 mM L-serine and 0.25 mM pyruvate) was about (1)/(10) of that through SDH. Flux through GCS accounted for only several percent of the amount of L-serine metabolized. Relative contributions of SDH and SPT/AGT to gluconeogenesis from L-serine were evaluated in vivo based on the principle that 3H at the 3 position of L-serine is mostly removed in the SDH pathway, whereas it is largely retained in the SPT/AGT pathway. The results showed that SPT/AGT contributed only 10-20% even after the enhancement of its activity by glucagon. These results suggested that SDH is the major metabolic exit of L-serine in rat liver. (+info)
Substitution of pyridoxal 5'-phosphate in D-serine dehydratase from Escherichia coli by cofactor analogues provides information on cofactor binding and catalysis.
D-Serine dehydratase (DSD) is a pyridoxal 5'-phosphate-dependent enzyme that catalyzes the conversion of D-serine to pyruvate and ammonia. Spectral studies of enzyme species where the natural cofactor was substituted by pyridoxal 5'-sulfate (PLS), pyridoxal 5-deoxymethylene phosphonate (PDMP), and pyridoxal 5'-phosphate monomethyl ester (PLPMe) were used to gain insight into the structural basis for binding of cofactor and substrate analogues. PDMP-DSD exhibits 35% of the activity of the native enzyme, whereas PLS-DSD and PLPMe-DSD are catalytically inactive. The emission spectrum of native DSD when excited at 280 nm shows maxima at 335 and 530 nm. The energy transfer band at 530 nm is very likely generated as a result of the proximity of Trp-197 to the protonated internal Schiff base. The cofactor analogue-reconstituted DSD species exhibit emission intensities decreasing from PLS-DSD, to PLPMe-DSD, and PDMP-DSD, when excited at 415 nm. Large increases in fluorescence intensity at 530 (540) nm can be observed for cofactor analogue-reconstituted DSD in the presence of substrate analogues when excited at 415 nm. In the absence and presence of substrate analogues, virtually identical far UV CD spectra were obtained for all DSD species. The visible CD spectra of native DSD, PDMP-DSD, and PLS-DSD exhibit a band centered on the visible absorption maximum with nearly identical intensity. Addition of substrate analogues to native and cofactor analogue-reconstituted DSD species results in most cases in a decrease or elimination of ellipticity. The results are interpreted in terms of local conformational changes and/or changes in the orientation of the bound cofactor (analogue). (+info)
Escherichia coli K-12 mutant forming a temperature-sensitive D-serine deaminase.
A single-site mutant of Escherichia coli K-12 able to grow in minimal medium in the presence of D-serine at 30 C but not at 42 C was isolated. The mutant forms a D-serine deaminase that is much more sensitive to thermal denaturation in vitro at temperatures above but not below 47 C than that of the wild type. No detectable enzyme is formed by the mutant at 42 C, however, and very little is formed at 37 C. The mutant enzyme is probably more sensitive to intracellular inactivation at high temperatures than the wild-type enzyme. The mutation lies in the dsdA region. The mutant also contains a dsdO mutation, which does not permit hyperinduction of D-serine deaminase synthesis. (+info)
Isolation and characterization of D-serine deaminase constitutive mutants by utilization of D-serine as sole carbon or nitrogen source.
Mutants constitutive for D-serine deaminase (Dsdase) synthesis were isolated by utilizing D-serine as sole nitrogen or carbon source in the chemostat. This method generated only regulatory constitutive (dsdC) mutants. The altered dsdC gene product in these strains is apparently able to bind D-serine more efficiently than the wild-type dsdC+ gene product--a selective advantage. Constitutive synthesis of Dsdase in all of these dsdC mutants is extremely sensitive to catabolite repression, and catabolite repression is reversed by the addition of D-serine. Of the 15 mutants generated by this method, none are suppressible by supD, supE, or supF. Mutations to a low level of constitutivity (maximal specific activity of 9) occur much more frequently than mutations to a high level (maximal specific activity of 79). High level constitutive synthesis of Dsdase results from the synthesis of an altered dsdC gene product--not from loss of ability to form the dsdC product. Dsdase synthesis is not regulated by the nitrogen supply in the medium, as nitrogen starvation does not result in the derepression of Dsdase synthesis. (+info)
Positive control in the D-serine deaminase system of Escherichia coli K-12.
Two new types of D-serine deaminase (Dsdase)-negative mutants have been isolated and characterized. The first fails to synthesize a functional dsdC gene product as a result of dsdC- (regulator negative) mutations. The mutations lie in the dsdC region, are cis and trans recessive to dsdC+, and give rise to revertants of novel regulatory phenotype. The second class consists of Dsdase-negative lysogens in which the phenotype is the result of the integration of lambdac1857 Sam7 into the dsdC region. Lambda lysates derived from two of the Dsdase-negative lysogens can transduce the structural gene for Dsdase (dsdA) but not the dsdC region. The dsdC+ gene product had no repressor effect on constitutive synthesis in a strain containing a dsdO (initiator constitutive) and a dsdC- mutation. These and other findings indicate that control of Dsdase synthesis is strictly positive. The partial trans effect of the dsdC+ gene product on constitutive synthesis in dsdCc (regulator constitutive) strains can thus be explained by "subunit mixing" between active dsdCc subunits and dsdC+ subunits which are inactive in the absence of the inducer, D-serine. The order of genes in the dsd region is supN-dsdC-dsdP-dsdA-aroC. (+info)
Resolution of D-serine dehydratase by cysteine. An analytical treatment.
A general method is presented for analysis of the resolution of pyridoxal-P-requiring enzymes by carbonyl reagents. The method is useful for accurately determining the very small equilibrium constants (KP) which characterize the dissociation of cofactor from many pyridoxal-P-requiring enzymes. The analysis also establishes the minimum number and relative stabilities of distinct enzymic species involved in the resolution process. Analysis of the resolution of D-serine dehydratase by L-and D-cysteine resulted in the establishment of an enzyme bound thiazolidine derivative as an intermediate in the pathway for resolution. The over-all equilibrium constant (KR) for the reaction, D-serine dehydratase + cystein in equilibrium KR thiazolidine derivative +D-serine apodehydratase was determined. At pH 7.80, T/2 0.33, 25 degrees, KR equal to 1.08 times 10-minus 3. A value of 7.0 nM for the equilibrium constant for the dissociation of D-serine dehydratase to apoenzyme and free pyridoxal-P was determined from the ratio KR/KT, where KT is the equilibrium constant for the formation of a thiazolidine derivative from free pyridoxal-P and cysteine. An estimate of 14 nM for KP was also obtained from partial resolution of D-serine dehydratase by high dilution. The difficulties associated with this direct determination of KP from the dependence on the enzyme concentration of the activity of very dilute solutions of enzyme are discussed. (+info)
Enhanced level and metabolic regulation of methionyl-transfer ribonucleic acid synthetase in different strains of Escherichia coli K-12.
The methionyl-transfer ribonucleic acid (tRNA) synthetase of Escherichia coli K-12 eductants carrying P2-mediated deletions in the region of the structural gene of this enzyme was investigated. No structural alteration of this enzyme was observed in three eductants examined. These were isolated from strain AB311, which had a threefold higher level of methionyl-tRNA synthetase than most haploid strains examined. In two of the three eductants studied, the level of this enzyme was twofold higher than in their parental strain regardless of growth conditions used. In contrast, isoleucyl-, leucyl-, and valyl-tRNA synthetases had similar levels in all strains examined. Like valyl-tRNA synthetase, but to a lesser extent, methionyl-tRNA synthetase was subject to metabolic regulation. Coupling between the level of methionyl-tRNA synthetase and growth rate was observed even in strains that had an enhanced level of methionyl-tRNA synthetase. These results suggest that the formation of methionyl-tRNA synthetase remains subject to metabolic regulation even when the repression-like mechanism that controls the synthesis of this enzyme is altered. In addition, we report that in the merodiploid strain EM20031, which was haploid for the valyl-tRNA synthetase structural gene and diploid for the structural genes of methionyl-tRNA synthetase and D-serine deaminase, the levels of these latter two enzymes varied to a minor yet significant extent with the phosphate concentration of the culture medium; under the same conditions, the level of valyl-tRNA synthetase remained unchanged. Moreover, no variation of the levels of these three enzymes in response to phosphate was observed in the haploid strain HfrH. These results indicate that in the merodiploid strain EM20031, which carries the episome F32, the number of episomes per chromosome varies to some extent according to the phosphate concentration of the culture medium. (+info)