Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group

Jagath, J.R; Rao, N. Appaji; Savithri, H.S.

doi:10.1042/bj3270877

Cited by 18 publications

(19 citation statements)

References 32 publications

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“…Similarly, it was shown in eSHMT that the conserved T226 plays an important role in converting geminal diamine into the external aldimine [17]. 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].…”

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%

“…rSHMT (wild type) and D89N SHMT were overexpressed and purified as described previously [19]. Briefly, the purification procedure involved ammonium sulphate fractionation, CMSephadex chromatography followed by gel-filtration using a Sephacryl S-200 column.…”

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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“…These mutants had significantly lower specific activity. Mutants such as K256Q, K256R and R401A were inactive [7,10]. The residue(s) involved in H % -folate binding have not been clearly defined by site-directed mutagenesis.…”

Section: Discussionmentioning

confidence: 99%

“…In contrast, the active-site lysine residue, Lys-229, in eSHMT participates in product expulsion [8]. An analysis of mutants led to the identification of Arg-363 in eSHMT [9] and Arg-401 in rSHMT [10] (residues equivalent to each other) as being involved in interacting with the α-carboxy group of serine.…”

Section: Introductionmentioning

confidence: 99%

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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“…Molecular Interaction between the Guanido Group and the Carboxyl Group-Molecular interaction between the guanido group of arginine and the carboxyl group in the substrate or the active site residues of enzyme was reported to be crucially involved in the interaction between the substrate and the enzyme (31)(32)(33)(34)(35)(36). The ionized guanido group of arginine can form a planar structure due to the sp 2 configuration of guanido carbon atom, so that a stable planar ion pair can be generated by forming a coplanar structure between the guanido group and the negatively charged carboxyl group.…”

Section: Figure 3 Stereo View Of Mae2 In Complex With Malonate or Mamentioning

confidence: 99%

Arg-158 Is Critical in Both Binding the Substrate and Stabilizing the Transition-state Oxyanion for the Enzymatic Reaction of Malonamidase E2

Yun¹,

Lee²,

Shin³

et al. 2006

Journal of Biological Chemistry

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Malonamidase E2 (MAE2) from Bradyrhizobium japonicum is an enzyme that hydrolyzes malonamate to malonate and has a Ser-cis-Ser-Lys catalytic triad at the active site. The crystal structures of wild type and mutant MAE2 exhibited that the guanido group of Arg-158 could be involved in the binding of malonamate in which the negative charge of the carboxyl group could destabilize a negatively charged transition-state oxyanion in the enzymatic reaction. In an attempt to elucidate the specific roles of Arg-158, site-directed mutants, R158Q, R158E, and R158K, were prepared (see Table 1). The crystal structure of R158Q determined at 2.2 Å resolution showed that the guanido group of Arg-158 was important for the substrate binding with the marginal structural change upon the mutation. The k cat value of R158Q significantly decreased by over 1500-fold and the catalytic activity of R158E could not be detected. The k cat value of R158K was similar to that of the wild type with the K m value drastically increased by 100-fold, suggesting that Lys-158 of R158K can stabilize the negative charge of the carboxylate in the substrate to some extent and contribute to the stabilization of the transition-state oxyanion, but a single amine group of Lys-158 in R158K could not precisely anchor the carboxyl group of malonamate compared with the guanido group of Arg-158. Our kinetic and structural evidences demonstrate that Arg-158 in MAE2 should be critical to both binding the substrate and stabilizing the transition-state oxyanion for the catalytic reaction of MAE2.Serine-nucleophile hydrolase is one of the most widespread enzymes in biological system and includes proteases, esterases, and lipases of which the catalytic triads or dyads play a critical role in the enzymatic catalysis (1). In most cases, enzymatic hydrolysis involves the nucleophilic attack on the electrophilic carbonyl carbon in the substrate, resulting in the formation of an oxyanion. The oxyanion can be recognized and stabilized by the oxyanion hole for the rate enhancement of the enzymatic reaction (2). Consequently, the stabilization of the transition-state oxyanion is very important for the efficient catalysis in the serine-nucleophile hydrolase.An enzyme family, designated as amidase signature (AS) 2 enzymes, utilizes a novel Ser-cis-Ser-Lys catalytic triad that is distinctly different from the classical catalytic triad or its variant (3-5). The unique nomenclature of AS is attributed to the AS sequence including the conserved sequence of about 130 amino acids rich in glycine and serine (Fig. 1A) (6, 7). The AS enzyme family includes over 200 different enzymes in many different organisms. The major role of AS enzymes has been known to be the hydrolysis of the amide bond. The enzymatic reactions of AS enzymes are involved in many critical biological functions such as the catabolism of neuromodulatory fatty acid amide by fatty acid amide hydrolase (8 -11), the formation of Gln-tRNA Gln by aminotransferase (12, 13), the formation of indole-3-acetic acid, which plays a r...

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Role of Arg-401 of cytosolic serine hydroxymethyltransferase in subunit assembly and interaction with the substrate carboxy group

Cited by 18 publications

References 32 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

Arg-158 Is Critical in Both Binding the Substrate and Stabilizing the Transition-state Oxyanion for the Enzymatic Reaction of Malonamidase E2

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