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. 2018 Oct 1;74(Pt 10):617-624.
doi: 10.1107/S2053230X18011895. Epub 2018 Sep 19.

Structure of glyoxysomal malate dehydrogenase (MDH3) from Saccharomyces cerevisiae

Shu Moriyama  1 , Kazuya Nishio  1 , Tsunehiro Mizushima  1
Affiliations

Structure of glyoxysomal malate dehydrogenase (MDH3) from Saccharomyces cerevisiae

Shu Moriyama et al. Acta Crystallogr F Struct Biol Commun. .

Abstract

Malate dehydrogenase (MDH), a carbohydrate and energy metabolism enzyme in eukaryotes, catalyzes the interconversion of malate to oxaloacetate (OAA) in conjunction with that of nicotinamide adenine dinucleotide (NAD+) to NADH. Three isozymes of MDH have been reported in Saccharomyces cerevisiae: MDH1, MDH2 and MDH3. MDH1 is a mitochondrial enzyme and a member of the tricarboxylic acid cycle, whereas MDH2 is a cytosolic enzyme that functions in the glyoxylate cycle. MDH3 is a glyoxysomal enzyme that is involved in the reoxidation of NADH, which is produced during fatty-acid β-oxidation. The affinity of MDH3 for OAA is lower than those of MDH1 and MDH2. Here, the crystal structures of yeast apo MDH3, the MDH3-NAD+ complex and the MDH3-NAD+-OAA ternary complex were determined. The structure of the ternary complex suggests that the active-site loop is in the open conformation, differing from the closed conformations in mitochondrial and cytosolic malate dehydrogenases.

Keywords: MDH3; Saccharomyces cerevisiae; X-ray crystallography; fatty-acid β-oxidation; glyoxysome; malate dehydrogenase.

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Figures

Figure 1
Figure 1
MDH3 regenerates NAD+ by reducing OAA to malate during fatty-acid β-oxidation. The glyoxysomal membrane is impermeable to large molecules such as NAD+. NAD+ is supplied from intra-glyoxysomal NADH by MDH3.
Figure 2
Figure 2
Amino-acid sequence alignment around the active-site loop and active-site residues in yeast MDHs and those from other organisms. The secondary-structure elements and sequence numbering correspond to those of MDH3. The active-site loop and active-site residues are shown in cyan and green boxes, respectively. Multiple sequence alignment was performed using ClustalW (Thompson et al., 1994 ▸). The secondary-structure assignment and figure were produced using ESPript (Robertson, 2014 ▸). Cl, Citrullus lanatus; Hs, Homo sapiens; Ss, Sus scrofa; g, glyoxysomal; m, mitochondrial; c, cytosomal.
Figure 3
Figure 3
Structures of yeast apo MDH3 and the MDH3–NAD+ and MDH3–NAD+–OAA complexes. (a) Dimeric apo MDH3 with each monomer (subunits A and B) highlighted in a different color. α-Helices are labeled α1–α14, β-strands are labeled β1–β11, 310-helices are labeled η1–η5 and the N-terminal NAD+-binding domain is indicated in light colors. (b) Conservation of the amino-acid residues involved in the interaction between subunits A and B of the MDH3 dimer. Amino-acid residues contributing to interactions are represented by stick models. A total of 44 sequences of all types of MDH (mitochondrial, glyoxysomal and cytosolic) were analyzed using the ConSurf server (Ashkenazy et al., 2010 ▸). The conservation grades of amino-acid residues are indicated using a color-coded scale from turquoise to maroon. The residues with the highest and lowest conservation score in the interaction surface are labeled in red and blue, respectively. The sequences used for ConSurf analysis are listed in Supplementary Table S1. (c) Structure of the MDH3–NAD+ complex. (d) Structure of the MDH3–NAD+–OAA complex. NAD+ and OAA are shown as yellow and magenta sticks, respectively.
Figure 4
Figure 4
Stereoviews of the NAD+- and OAA-binding sites of MDH3. Close-up views of NAD+-bound MDH3 (a) and NAD+/OAA-bound MDH3 (b) are shown. NAD+ and OAA were modeled using an F o − F c OMIT electron-density map contoured at 3σ. Residues that are in contact with or form hydrogen bonds to NAD+ and OAA are indicated by stick models and dashed lines, respectively.
Figure 5
Figure 5
Comparison of the open and closed conformations of the active-site loops in NAD+/OAA-bound MDHs. Stereoview of the active sites of human mitochondrial MDH (PDB entry 4wlo, black) and yeast MDH3 (gray). Active-site loops and ligands of human mitochondrial MDH and yeast MDH3 are highlighted in magenta and green, respectively. NAD+, OAA and highly conserved active-site residues are indicated using stick models.
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