[feed] Atom [feed] RSS 1.0 [feed] RSS 2.0

Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn341, Tyr60 and Phe351 in tetrahydrofolate binding

Vinitha, R. Pai and Rajaram, V. and Shveta, Bisht and Bhavani, B. S. and Appaji Rao, N. and Murthy, M. R. N. (2009) Structural and functional studies of Bacillus stearothermophilus serine hydroxymethyltransferase: the role of Asn341, Tyr60 and Phe351 in tetrahydrofolate binding. Biochemical Journal, 418 (3). pp. 635-642.

[img] PDF
Biochem,_J_418(3)_2009_635.pdf
Restricted to Registered users only

Download (306kB)

Abstract

SHMT (serine hydoxymethyltransferase), a type I pyridoxal 5'- phosphate-dependent enzyme, catalyses the conversion of L-serine and THF (tetrahydrofolate) into glycine and 5,10-methylene THF. SHMT also catalyses several THF-independent side reactions such as cleavage of β-hydroxy amino acids, transamination, racemization and decarboxylation. In the present study, the residues Asn341, Tyr60 and Phe351, which are likely to influence THF binding, were mutated to alanine, alanine and glycine respectively, to elucidate the role of these residues in THFdependent and -independent reactions catalysed by SHMT. The N341A and Y60A bsSHMT (Bacillus stearothermophilus SHMT) mutants were inactive for the THF-dependent activity, while the mutations had no effect on THF-independent activity. However, mutation of Phe351 to glycine did not have any effect on either of the activities. The crystal structures of the glycine binary complexes of the mutants showed that N341A bsSHMT forms an external aldimine as in bsSHMT, whereas Y60A and F351G bsSHMTs exist as a mixture of internal/external aldimine and gem-diamine forms. Crystal structures of all of the three mutants obtained in the presence of L-allo-threonine were similar to the respective glycine binary complexes. The structure of the ternary complex of F351G bsSHMT with glycine and FTHF (5- formyl THF) showed that the monoglutamate side chain of FTHF is ordered in both the subunits of the asymmetric unit, unlike in the wild-type bsSHMT. The present studies demonstrate that the residues Asn341 and Tyr60 are pivotal for the binding of THF/FTHF, whereas Phe351 is responsible for the asymmetric binding of FTHF in the two subunits of the dimer.

Item Type: Article
Uncontrolled Keywords: L-allo-threonine, crystal structure, folate binding, 5- formyl tetrahydrofolate (FTHF), pyridoxal 5'-phosphate, serine hydroxymethyltransferase (SHMT), ternary complex
Subjects: 500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 26 Jun 2009 04:13
Last Modified: 28 Dec 2011 10:10
URI: http://ir.cftri.com/id/eprint/9110

Actions (login required)

View Item View Item