Serine hydroxymethyltransferase (SHMT), a pyridoxal 5′-phosphate (PLP)-dependent enzyme, catalyses the transfer of the hydroxymethyl group from serine to tetrahydrofolate to yield glycine and N5,N10-methylenetetrahydrofolate. An analysis of the known SHMT sequences indicated that several amino acid residues were conserved. In this paper, we report the identification of the amino acid residues essential for maintaining the oligomeric structure of sheep liver cytosolic recombinant SHMT (scSHMT) through intra- and inter-subunit interactions and by stabilizing the binding of PLP at the active site. The mutation of Lys-71, Arg-80 and Asp-89, the residues involved in intra-subunit ionic interactions, disturbed the oligomeric structure and caused a loss of catalytic activity. Mutation of Trp-110 to Phe was without effect, while its mutation to Ala resulted in the enzyme being present in the insoluble fraction. These results suggested that Trp-110 located in a cluster of hydrophobic residues was essential for proper folding of the enzyme. Arg-98 and His-304, residues involved in the inter-subunit interactions, were essential for maintaining the tetrameric structure. Mutation of Tyr-72, Asp-227 and His-356 at the active site which interact with PLP resulted in the loss of PLP, and hence loss of tetrameric structure. Mutation of Cys-203, located away from the active site, weakened PLP binding indirectly. The results demonstrate that in addition to residues involved in inter-subunit interactions, those involved in PLP binding and intra-subunit interactions also affect the oligomeric structure of scSHMT.
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February 2003
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Research Article|
February 01 2003
Identification of amino acid residues, essential for maintaining the tetrameric structure of sheep liver cytosolic serine hydroxymethyltransferase, by targeted mutagenesis
Venkatakrishna Rao JALA;
Venkatakrishna Rao JALA
1
Department of Biochemistry, Indian Institute of Science, Bangalore-560 012, India
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Naropantul APPAJI RAO;
Naropantul APPAJI RAO
Department of Biochemistry, Indian Institute of Science, Bangalore-560 012, India
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Handanahal Subbarao SAVITHRI
Handanahal Subbarao SAVITHRI
2
Department of Biochemistry, Indian Institute of Science, Bangalore-560 012, India
2To whom correspondence should be addressed (e-mail bchss@biochem.iisc.ernet.in).
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Publisher: Portland Press Ltd
Received:
July 23 2002
Revision Received:
October 04 2002
Accepted:
October 22 2002
Accepted Manuscript online:
October 22 2002
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London ©2003
2003
Biochem J (2003) 369 (3): 469–476.
Article history
Received:
July 23 2002
Revision Received:
October 04 2002
Accepted:
October 22 2002
Accepted Manuscript online:
October 22 2002
Citation
Venkatakrishna Rao JALA, Naropantul APPAJI RAO, Handanahal Subbarao SAVITHRI; Identification of amino acid residues, essential for maintaining the tetrameric structure of sheep liver cytosolic serine hydroxymethyltransferase, by targeted mutagenesis. Biochem J 1 February 2003; 369 (3): 469–476. doi: https://doi.org/10.1042/bj20021160
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