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Arg-265: a critical residue of L.donovani cytosolic SHMT in maintaining the binding of THF and catalysis.

Experimental parasitology (2014-12-17)
Shashi Gandhi, Neha Gaur, Shagun Krishna, Mohd Imran Siddiqi, Jitendra Kumar Saxena
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.

MATERIALS
Product Number
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Product Description

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