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Structure-function relationship in serine hydroxymethyltransferase.

N Appaji Rao1, M Ambili, Venkatakrishna R Jala

  • 1Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India.

Biochimica Et Biophysica Acta
|April 11, 2003
PubMed
Summary
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Serine hydroxymethyltransferase (SHMT) enzyme structure and function were investigated using mutagenesis. Key residues were identified for maintaining SHMT

Area of Science:

  • Biochemistry
  • Enzymology
  • Structural Biology

Background:

  • Serine hydroxymethyltransferase (SHMT) is a crucial enzyme in folate metabolism, utilizing pyridoxal-5'-phosphate (PLP) and tetrahydrofolate (H(4)-folate).
  • SHMT catalyzes the reversible conversion of serine to glycine, a reaction vital for one-carbon metabolism.
  • Understanding the structure-function relationship of SHMT is essential for elucidating its catalytic mechanisms and potential therapeutic targets.

Purpose of the Study:

  • To investigate the structure-function relationship of serine hydroxymethyltransferase (SHMT) through site-directed mutagenesis.
  • To identify key amino acid residues involved in SHMT's oligomeric structure, catalytic activity, and reaction mechanism.
  • To provide evidence for the reaction mechanism of SHMT, distinguishing between direct transfer and retro-aldol cleavage pathways.

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Main Methods:

  • Site-directed mutagenesis was employed to alter conserved and active site residues of SHMT.
  • Characterization of mutant enzymes, including analysis of oligomeric structure and catalytic activity.
  • Integration of mutational data with structural information of SHMT and its complexes.

Main Results:

  • Specific residues (K71, Y72, R80, D89, W110, S202, C203, H304, H306, H356) were identified as critical for maintaining the oligomeric structure of SHMT.
  • Mutation of D227 resulted in inactive dimers, highlighting its role in tetrameric stability and catalysis.
  • Residue E74 is involved in conformational changes (open to closed) rather than direct proton abstraction, while Y82 stabilizes the quinonoid intermediate.
  • K256 is crucial for Schiff base formation with PLP and tetrameric structure maintenance.
  • R262 mutations underscored the importance of distal interactions in catalysis.
  • Mutational analysis provided evidence supporting a direct transfer mechanism over retro-aldol cleavage for SHMT.

Conclusions:

  • The study elucidates the roles of specific amino acid residues in maintaining SHMT's quaternary structure and catalytic function.
  • Key residues have been identified that are critical for enzyme stability, substrate binding, and intermediate stabilization.
  • Evidence supports a direct transfer mechanism for the reaction catalyzed by SHMT, refining our understanding of its enzymatic pathway.