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Arg-265: a critical residue of L.donovani cytosolic SHMT in maintaining the binding of THF and catalysis.
Exp Parasitol. 2015 Feb; 149:16-23.EP

Abstract

Serine hydroxymethyltransferase belongs to the class of pyridoxal-5-phosphate enzymes along with aspartate aminotransferase. To explore the function of residue(s) involved in binding of the carboxylate group of Tetrahydrofolic acid (THF) to L. donovani cytosolic serine hydroxymethyltransferase (LdcSHMT), the gene was cloned in pET-28(a) vector, overexpressed and purified to homogeneity. With the help of docking results of THF to the active site of protein, the key residues involved in interaction were identified. In an attempt to unravel the function of Arg265 residue involved in binding of the carboxylate group of THF, Arg-265 was mutated to Ala by site-directed mutagenesis. The Arg265Ala-LdcSHMT showed increased Km value (threefold) and decreased kcat/Km value (threefold) for H₄-folate as compared with wild type enzyme. The wild and mutant enzymes exhibited similar Km and kcat/Km values for L-allo-threonine. Unlike the wild type enzyme, mutant failed to form characteristic quinonoid intermediate and was unable to carry out the exchange of α-proton from glycine in the presence of Tetrahydrofolate. These results suggested that Arg265 residue is required for the binding of Tetrahydrofolate and may be the base that abstracts α-proton from glycine, leading to formation of quinonoid intermediate in cytosolic SHMT of L. donovani.

Authors+Show Affiliations

Division of Biochemistry, Central Drug Research Institute, CSIR, Lucknow 226031, India.Division of Biochemistry, Central Drug Research Institute, CSIR, Lucknow 226031, India.Division of Molecular and Structural Biology, Central Drug Research Institute, CSIR, Lucknow 226031, India.Division of Molecular and Structural Biology, Central Drug Research Institute, CSIR, Lucknow 226031, India.Division of Biochemistry, Central Drug Research Institute, CSIR, Lucknow 226031, India. Electronic address: jkscdri@yahoo.com.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

25499510

Citation

Gandhi, Shashi, et al. "Arg-265: a Critical Residue of L.donovani Cytosolic SHMT in Maintaining the Binding of THF and Catalysis." Experimental Parasitology, vol. 149, 2015, pp. 16-23.
Gandhi S, Gaur N, Krishna S, et al. Arg-265: a critical residue of L.donovani cytosolic SHMT in maintaining the binding of THF and catalysis. Exp Parasitol. 2015;149:16-23.
Gandhi, S., Gaur, N., Krishna, S., Siddiqi, M. I., & Saxena, J. K. (2015). Arg-265: a critical residue of L.donovani cytosolic SHMT in maintaining the binding of THF and catalysis. Experimental Parasitology, 149, 16-23. https://doi.org/10.1016/j.exppara.2014.12.004
Gandhi S, et al. Arg-265: a Critical Residue of L.donovani Cytosolic SHMT in Maintaining the Binding of THF and Catalysis. Exp Parasitol. 2015;149:16-23. PubMed PMID: 25499510.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Arg-265: a critical residue of L.donovani cytosolic SHMT in maintaining the binding of THF and catalysis. AU - Gandhi,Shashi, AU - Gaur,Neha, AU - Krishna,Shagun, AU - Siddiqi,Mohd Imran, AU - Saxena,Jitendra Kumar, Y1 - 2014/12/09/ PY - 2014/05/04/received PY - 2014/11/21/revised PY - 2014/12/04/accepted PY - 2014/12/16/entrez PY - 2014/12/17/pubmed PY - 2015/3/25/medline KW - Cytosolic serinehydroxymethyltransferase KW - Leishmania donovani KW - Pyridoxal-5-phosphate KW - Site directed mutagenesis KW - Tetra hydrofolate SP - 16 EP - 23 JF - Experimental parasitology JO - Exp Parasitol VL - 149 N2 - Serine hydroxymethyltransferase belongs to the class of pyridoxal-5-phosphate enzymes along with aspartate aminotransferase. To explore the function of residue(s) involved in binding of the carboxylate group of Tetrahydrofolic acid (THF) to L. donovani cytosolic serine hydroxymethyltransferase (LdcSHMT), the gene was cloned in pET-28(a) vector, overexpressed and purified to homogeneity. With the help of docking results of THF to the active site of protein, the key residues involved in interaction were identified. In an attempt to unravel the function of Arg265 residue involved in binding of the carboxylate group of THF, Arg-265 was mutated to Ala by site-directed mutagenesis. The Arg265Ala-LdcSHMT showed increased Km value (threefold) and decreased kcat/Km value (threefold) for H₄-folate as compared with wild type enzyme. The wild and mutant enzymes exhibited similar Km and kcat/Km values for L-allo-threonine. Unlike the wild type enzyme, mutant failed to form characteristic quinonoid intermediate and was unable to carry out the exchange of α-proton from glycine in the presence of Tetrahydrofolate. These results suggested that Arg265 residue is required for the binding of Tetrahydrofolate and may be the base that abstracts α-proton from glycine, leading to formation of quinonoid intermediate in cytosolic SHMT of L. donovani. SN - 1090-2449 UR - https://www.unboundmedicine.com/medline/citation/25499510/Arg_265:_a_critical_residue_of_L_donovani_cytosolic_SHMT_in_maintaining_the_binding_of_THF_and_catalysis_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S0014-4894(14)00274-4 DB - PRIME DP - Unbound Medicine ER -