The Role of His-134, -147, and -150 Residues in Subunit Assembly, Cofactor Binding, and Catalysis of Sheep Liver Cytosolic Serine Hydroxymethyltransferase

Junutula, Jagath R.; Sharma, Balasubramanya; Rao, N. Appaji; Savithri, H.S.

doi:10.1074/jbc.272.39.24355

Cited by 45 publications

(52 citation statements)

References 48 publications

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“…It was shown that R363 in eSHMT and the corresponding residue R401 in rSHMT were essential for binding of substrate carboxy group [18,19]. Mutation of the conserved H134, H147 and H150 in rSHMT established their roles in the maintenance of oligomeric structure, cofactor binding and proton abstraction respectively [20]. Furthermore, the N-terminal arm was shown to be important for stabilizing the tetrameric structure of rSHMT [21].…”

Section: Introductionmentioning

confidence: 99%

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Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

et al. 1999

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Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5h-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 µM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5 % active. The

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Section: Introductionmentioning

confidence: 99%

“…The dialysed enzyme was used for further studies. Protein was estimated by measuring the absorbance at 280 nm, 1 absorbance unit was equivalent to 1n2 mg\ml [20].…”

Section: Purification Of Wild-type and D89n Shmtmentioning

confidence: 99%

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

et al. 1999

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“…The K m values for -serine and H % -folate were slightly higher than those obtained for rSHMT, suggesting that substrate binding was not significantly affected. Similarly, the K m values for -serine for H134N, H147N, H230Y and D89N mutant SHMTs were also not significantly altered and were 0n92, 1n9, 1n1 and 0n9 mM respectively, compared with 1 mM for rSHMT [22,30,31]. These mutants had significantly lower specific activity.…”

Section: Discussionmentioning

confidence: 88%

“…The kinetic parameters for -serine and H % -folate and the aldolytic cleavage of -allothreonine were monitored as described by Jagath et al [22]. The rates of transamination of -alanine were determined as described previously [23].…”

Section: Enzyme Assaysmentioning

confidence: 99%

“…It was shown previously that the mutation of residues of rSHMT involved in interaction with PLP led to the dissociation of the tetrameric structure of the protein [7,22]. The observation that P297R had decreased enzyme activity, although it contained stoichiometric amounts of PLP, prompted an examination of the effect of the removal and re-addition of PLP on the oligomeric status of the protein.…”

Section: Oligomeric Status Of the Mutant Proteinmentioning

confidence: 99%

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Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Talwar

Leelavathy

Rao

et al. 2000

Biochemical Journal

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Serine hydroxymethyltransferase belongs to the α class of pyridoxal-5´-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the γ-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85–90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5´-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with l-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.

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Serine hydroxymethyltransferase from the silkworm Bombyx mori: Identification, distribution, and biochemical characterization

Haque

Hirowatari

Nai

et al. 2019

Arch Insect Biochem Physiol

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Serine hydroxymethyltransferase (SHMT) catalyzes the interconversion of serine and tetrahydrofolate (THF) to glycine and methylenetetrahydrofolate. cDNA encoding Bombyx mori SHMT (bmSHMT) was cloned and sequenced. The deduced amino acid sequence consisted of 465 amino acids and was found to share homology with other SHMTs. Recombinant bmSHMT was overexpressed in Escherichia coli and purified to homogeneity. The enzyme showed optimum activity at pH 3.0 and 30°C and was stable under acidic conditions. The Km and kcat/Km values for THF in the presence of Nicotinamide adenine dinucleotide phosphate (NADP+) were 0.055 mM and 0.081 mM−1 s−1, respectively, whereas those toward NADP+ were 0.16 mM and 0.018 mM−1 s−1 and toward l‐serine were 1.8 mM and 0.0022 mM−1 s−1, respectively. Mutagenesis experiments revealed that His119, His132, and His135 are important for enzymatic activity. Our results provide insight into the roles and regulation mechanism of one‐carbon metabolism in the silkworm B. mori.

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The Role of His-134, -147, and -150 Residues in Subunit Assembly, Cofactor Binding, and Catalysis of Sheep Liver Cytosolic Serine Hydroxymethyltransferase

Abstract: In an attempt to unravel the role of conserved histidine residues in the structure-function of sheep liver cytosolic serine hydroxymethyltransferase (SHMT), three site-specific mutants (H134N, H147N, and H150N

Cited by 45 publications

References 48 publications

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Serine hydroxymethyltransferase from the silkworm Bombyx mori: Identification, distribution, and biochemical characterization

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