Crystal structure of serine dehydratase from rat liver

SDH (L-serine dehydratase, EC 4.3.1.17) catalyzes the pyridoxal 5'-phosphate (PLP)-dependent dehydration of L-serine to yield pyruvate and ammonia. Liver SDH plays an important role in gluconeogenesis. Formation of pyruvate by SDH is a two-step reaction in which the hydroxyl group of serine is cleaved to produce aminoacrylate, and then the aminoacrylate is deaminated by nonenzymatic hydrolysis to produce pyruvate. The crystal structure of rat liver apo-SDH was determined by single isomorphous replacement at 2.8 A resolution. The holo-SDH crystallized with O-methylserine (OMS) was also determined at 2.6 A resolution by molecular replacement. SDH is composed of two domains, and each domain has a typical alphabeta-open structure. The active site is located in the cleft between the two domains. The holo-SDH contained PLP-OMS aldimine in the active site, indicating that OMS can form the Schiff base linkage with PLP, but the subsequent dehydration did not occur. Apo-SDH forms a dimer by inserting the small domain into the catalytic cleft of the partner subunit so that the active site is closed. Holo-SDH also forms a dimer by making contacts at the back of the clefts so that the dimerization does not close the catalytic cleft. The phosphate group of PLP is surrounded by a characteristic G-rich sequence ((168)GGGGL(172)) and forms hydrogen bonds with the amide groups of those amino acid residues, suggesting that the phosphate group can be protonated. N(1) of PLP participates in a hydrogen bond with Cys303, and similar hydrogen bonds with N(1) participating are seen in other beta-elimination enzymes. These hydrogen bonding schemes indicate that N(1) is not protonated, and thus, the pyridine ring cannot take a quinone-like structure. These characteristics of the bound PLP suggest that SDH catalysis is not facilitated by forming the resonance-stabilized structure of the PLP-Ser aldimine as seen in aminotransferases. A possible catalytic mechanism involves the phosphate group, surrounded by the characteristic sequence, acting as a general acid to donate a proton to the leaving hydroxyl group of serine.     

Periportal expression of the serine dehydratase gene in rat liver

Summary The mRNA for rat liver serine dehydratase, a gluconeogenic enzyme, exhibits a circadian rhythm with a maximum at the onset of darkness marking the end of the fasting period and a minimum at the onset of light that marks the end of the feeding period, when rats have free access to food and water.In situ hybridization with an antisense cRNA probe revealed that serine dehydratase mRNA was localized in the periportal area of rat liver parenchyma in the evening, whereas it was scarce in the liver in the morning. The predominant localization of serine dehydratase mRNA in the periportal area also occurred in livers of rats that underwent laparotomy, glucagon and dexamethasone administration, and streptozotocin-induced diabetes mellitus, all of which are known to induce serine dehydratase mRNA levels remarkably. Immunostaining revealed that the localization of serine dehydratase protein agreed with that of succinate dehydrogenase, another enzyme known to be predominant in the periportal zone. Thus, the periportal serine dehydratase gene expression strongly supports the idea of metabolic zonation that gluconeogenesis from amino acids occurs preferentially in the periportal parenchyma of rat liver.     

Evidence for a dimeric structure of rat liver serine dehydratase

Rat liver serine dehydratase (SDH) is known to be involved in gluconeogenesis. It has long been believed to be a dimeric protein with the subunit molecular weight (M(r)) of 34,000. Recently, sheep liver SDH was reported to be a monomer with a M(r) of 38,000. The native M(r) of rat SDH was only determined by the ultracentrifugation method more than three decades ago, and that of sheep SDH was done by the method of gel chromatography. The primary to quaternary structures of a given enzyme in a specific mammalian organ are usually conserved among various species. The aim of the present investigation is to clarify the structural differences between rat and sheep SDHs. First, we found that the amino acid composition reported for sheep SDH was statistically similar to that of rat SDH. Second, immunoblot analysis using anti-rat SDH IgG as the probe showed the size of sheep SDH to be a M(r) of 30,500, whereas that of SDH was about M(r) of 35,000. On the other hand, the native size of rat SDH was assessed by two methods: (1) the laser light scattering method demonstrated that rat SDH had a M(r) of 66,800, consistent with the previous value (M(r)=64,000); (2) cross-linking experiments of the purified rat SDH with dimethyl suberimidate revealed the existence of a dimeric form by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present results clearly confirm that rat SDH is a dimer, and suggest that sheep SDH is similar to rat SDH immunologically, but with a molecular weight 7500 smaller than reported previously.     

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3'- and 5'-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34,462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases was found as a highly conserved sequence.     

Developmental and growth-related regulation of expression of serine dehydratase mRNA in rat liver

In rat liver, serine dehydratase mRNA is undetectable in the late prenatal period, but its level increases rapidly after birth to a transient peak, and then after decrease gradually increases again to a maximum 2 weeks after birth that is slightly higher than that of adult liver. To determine whether mature quiescent hepatocytes proliferate without loss of differentiated functions, we measured the serine dehydratase mRNA contents in regenerating liver and primary cultured hepatocytes from adult rats. Partial hepatectomy resulted in a dramatic decrease in the mRNA content within 24 h and then its recovery within a week. In subconfluent cultures of adult rat hepatocytes that did not grow even in the presence of mitogens, serine dehydratase mRNA was maintained at a high level. However, when the hepatocytes were cultured at low cell density without added mitogens, their serine dehydratase mRNA content decreases to a quarter of that of subconfluent cultures. The possibility that the expression of serine dehydratase mRNA is regulated in G0/G1 transition before entry into the S phase and the relationship of the mRNA with growth are discussed.     

Molecular cloning of DNA complementary to mRNA of rat liver serine dehydratase

A cDNA clone containing sequences complementary to the mRNA cording for rat hepatic serine dehydratase was isolated to study the multihormonal regulation of this enzyme. Serine dehydratase mRNA was partially purified (50-fold enrichment, 8.2% of the total mRNA activity) from the liver of rats fed high protein diet by polysome immunoadsorption followed by oligo(dT)-cellulose column chromatography. This preparation was used as template for synthesis of cDNA. Double-stranded cDNA sequences were inserted into the plasmid pBR322 and cloned in Escherichia coli DH1. Of 860 transformants screened, 6 clones containing DNA complementary to serine dehydratase mRNA were identified by differential colony hybridization and hybrid-selected translation. The length of serine dehydratase mRNA was estimated to be 1,500 bases by Northern blot analysis. One cloned cDNA comprised about 1,000 base pairs, or 65% of the length of the mRNA. The amount of the mRNA was greatly increased in the liver of rats given high protein diet.     

Immunochemical studies of serine dehydratase and ornithine aminotransferase regulation in rat liver in vivo.

Previous studies of serine dehydratase (EC 4.2.1.13) and ornithine aminotransferase (EC 2.6.1.13) adaptation in rat liver showed that in rats on a high protein diet, glucocorticoid administration increased serine dehydratase activity while simultaneously reducing the activity of ornithine aminotransferase. The present study examines the role of enzyme synthesis in the expression of these and other dissimilar adaptive characteristics of the two enzymes. Both enzymes were purified to crystallinity and used to prepare specific antibodies. Changes in the rate of synthesis of each enzyme during adaptation were then measured immunochemically. In rats fed ad libitum, the synthetic rates for both enzymes exhibited circadian rhythm, although enzyme levels remained relatively constant. The circadian cycle for ornithine aminotransferase synthesis was in phase with the cycles for body weight and relative liver weight (maxima at 9 a.m., minima at 9 p.m.) but was approximately 12 hours out of phase with the cycle for serine dehydratase synthesis. 9alpha-Fluoro-11beta, 21-dihydroxy-16alpha, 17alpha-isopted at 9 a.m., increased serine dehydratase synthesis and simultaneously decreased the synthesis of ornithine aminotransferase. When triamcinolone was injected at 9 p.m., however, serine dehydratase synthesis was not stimulated, although the reduction of ornithine aminotransferase synthesis was still produced. These results suggest that: (a) circadian cycling of synthesis may be a general phenomenon in enzyme regulation even though for enzymes with relatively long half-lives, such cycling may not be reflected as fluctuations in enzyme levels; (b) such circadian rhythmicity may also involve cyclic changes in the responsiveness of the enzyme-forming system to regulatory stimuli; (c) whereas the adaptive behavior of serine dehydratase typifies that of amino acid-catabolizing enzymes in general, the responses of ornithine aminotransferase denote a functional association of this enzyme with anabolic processes. On this basis, the possibility that ornithine aminotransferase plays a pivotal role in the regulation of urea cycle activity and nitrogen balance is discussed.     

Response of the induction of rat liver serine dehydratase to changes in the dietary protein requirement

Growing and mature rats were examined for the effect of a change in dietary protein requirements on the induction of liver serine dehydratase (SDH). The rats were fed on diets varying in casein content, and the weight change and nitrogen balance was determined. SDH activity and its gene expression were induced in both growing and mature rats when their protein intake exceeded their nutritional requirements.     

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.