Asp-89: a critical residue in maintaining the oligomeric structure of sheep liver cytosolic serine hydroxymethyltransferase

Abstract

Aspartate residues function as proton acceptors in catalysis and are involved in ionic interactions stabilizing subunit assembly. In an attempt to unravel the role of a conserved aspartate (D89) in sheep-liver tetrameric serine hydroxymethyltransferase (SHMT), it was converted into aspargine by site-directed mutagenesis. The purified D89N mutant enzyme had a lower specific activity compared with the wild-type enzyme. It was a mixture of dimers and tetramers with the proportion of tetramers increasing with an increase in the pyridoxal-5h-phosphate (PLP) concentration used during purification. The D89N mutant tetramer was as active as the wild-type enzyme and had similar kinetic and spectral properties in the presence of 500 µM PLP. The quinonoid spectral intermediate commonly seen in the case of SHMT was also seen in the case of D89N mutant tetramer, although the amount of intermediate formed was lower. Although the purified dimer exhibited visible absorbance at 425 nm, it had a negligible visible CD spectrum at 425 nm and was only 5 % active. The

Metabolic control of BRISC–SHMT2 assembly regulates immune signalling

“…2c, d). This shift was largely reduced or abolished in A285T and K280A SHMT2 mutants, in agreement with studies showing SHMT2 mutations that abrogate PLP binding shift the equilibrium towards the dimer 1,16–19 (Extended Data Fig. 2e).…”