Identification of catalytically important amino acid residues for enzymatic reduction of glyoxylate in plants

Abstract

NADPH-dependent glyoxylate reductases from Arabidopsis thaliana (AtGLYR) convert both glyoxylate and succinic semialdehyde into their corresponding hydroxyacid equivalents. The primary sequence of cytosolic AtGLYR1 reveals several sequence elements that are consistent with the β-HAD (β-hydroxyacid dehydrogenase) protein family, whose members include 3-hydroxyisobutyrate dehydrogenase, tartronate semialdehyde reductase and 6-phosphogluconate dehydrogenase. Here, site-directed mutagenesis was utilized to identify catalytically important amino acid residues for glyoxylate reduction in AtGLYR1. Kinetic studies and binding assays established that Lys170 is essential for catalysis, Phe231, Asp239, Ser121 and Thr95 are more important in substrate binding than in catalysis, and Asn174 is more important in catalysis. The low activity of the mutant enzymes precluded kinetic studies with succinic semialdehyde. The crystal structure of AtGLYR1 in the absence of substrate was solved to 2.1Å by molecular replacement using a previously unrecognized member of the β-HAD family, cytokine-like nuclear factor, thereby enabling the 3-D structure of the protein to be modeled with substrate and co-factor. Structural alignment of AtGLYR1 with β-HAD family members provided support for the essentiality of Lys170, Phe173, Asp239, Ser121, Asn174 and Thr95 in the active site and preliminary support for an acid/base catalytic mechanism involving Lys170 as the general acid and a conserved active-site water molecule. This information established that AtGLYR1 is a member of the β-HAD protein family. Sequence and activity comparisons indicated that AtGLYR1 and the plastidial AtGLYR2 possess structural features that are absent in Arabidopsis hydroxypyruvate reductases and probably account for their stronger preference for glyoxylate over hydroxypyruvate.

Keywords: 3-HIBADH; 3-hydroxyisobutyrate dehydrogenase; 6-PGDH; 6-phosphogluconate dehydrogenase; Arabidopsis thaliana; Arabidopsis thaliana glyoxylate reductase; AtGLYR; Glyoxylate reductase; HPR; Hydroxypyruvate reductase; Photorespiration; SSA; Succinic semialdehyde reductase; TA; TSAR; hydroxypyruvate reductase; succinic semialdehyde; tartaric acid; tartronate semialdehyde reductase; β-HAD; β-hydroxyacid dehydrogenase; β-hydroxyacid dehydrogenase protein family.