Folate polyglutamylation eliminates dependence of activity on enzyme concentration in mitochondrial serine hydroxymethyltransferases from Arabidopsis thaliana

• Published in: Archives of biochemistry and biophysics Vol. 536; no. 1; pp. 87 - 96
• Main Authors: Wei, Zhaoyang, Sun, Kehan, Sandoval, Francisco J., Cross, Joanna M., Gordon, Christine, Kang, ChulHee, Roje, Sanja
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2013
• Subjects: Amino Acid Sequence; Arabidopsis - chemistry; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis - metabolism; Cloning, Molecular; Enzyme Activation; Folate polyglutamylation; Folic Acid - metabolism; Glycine Hydroxymethyltransferase - chemistry; Glycine Hydroxymethyltransferase - genetics; Glycine Hydroxymethyltransferase - metabolism; Kinetics; Mitochondria - chemistry; Mitochondria - enzymology; Mitochondria - genetics; Mitochondria - metabolism; Models, Molecular; Molecular Sequence Data; One-carbon metabolism; Peptides - metabolism; Protein Multimerization; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; Serine hydroxymethyltransferase (SHMT); Tetrahydrofolate; Tetrahydrofolates - metabolism; One-carbon metabolism; Folate polyglutamylation; Serine hydroxymethyltransferase (SHMT); Tetrahydrofolate
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2013.06.004

•Three recombinant SHMTs from A. thaliana (AtSHMTs) were characterized.•Two enzymes faster at higher enzyme concentrations with monoglutamate folates.•The effect absent in the presence of pentaglutamate folate substrates.•No evidence for a change in the oligomerization state. The reversible reaction catalyzed by serine hydroxymethyltransferase (SHMT) is the major one-carbon unit source for essential metabolic processes. The Arabidopsis thaliana genome encodes seven SHMT isozymes localized in mitochondria, plastids, nuclei, and the cytosol. Knowledge of the biochemical properties of each isozyme is central to understanding and manipulating one-carbon metabolism in plants. We heterologously expressed and purified three recombinant SHMTs from A. thaliana (AtSHMTs) putatively localized in mitochondria (two) and the cytosol (one). Their biochemical properties were characterized with respect to the impact of folate polyglutamylation on substrate saturation kinetics. The two mitochondrial AtSHMTs, but not the cytosolic one, had increased turnover rates at higher (>0.4ng/μL) enzyme concentrations in the presence of monoglutamylated folate substrates, but not in the presence of pentaglutamylated folate substrates. We found no experimental support for a change in oligomerization state over the range of enzyme concentration studied. Modeling of the enzyme structures presented features that may explain the activity differences between the mitochondrial and cytosolic isozymes.