Conformational changes on substrate binding revealed by structures of Methylobacterium extorquens malate dehydrogenase

Abstract

Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD⁺, and a ternary complex of MDH and oxaloacetate with ADP-ribose occupying the pyridine nucleotide-binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate-binding site is accessible in the enzyme resting state or NAD⁺-bound forms, the substrate-bound form exhibits a closed conformation. This conformational change involves the transition of an α-helix to a 3₁₀-helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)-malate.

Keywords: Methylobacterium extorquens; biofuels; malate dehydrogenase; methylotrophs.

Figure 1

Overall structure of MexMDH. The polypeptide displays a Rossmann fold of the form (βαβ)₂(αβ)₂, typical of NAD⁺-binding MDH/LDH-like proteins; the protein is arranged into a tetramer with one active site per monomer. The figure displays the MexMDH–NAD⁺ complex as obtained in this work (PDB entry 5ujk).

Figure 2

Binding of NAD⁺ and of OAA and APR by MexMDH. (a) The carboxamide group of NAD⁺ (blue) appears to be hydrogen-bonded to the Met144 and Ile119 backbone carbonyls through its amino group, and to the protonated His176 at N^∊2 through its carbonyl O atom. The Asp149 carboxylate also appears to be hydrogen-bonded to the protonated His176 at N^δ1. (b) Binding of OAA and APR (orange) induces a significant reorganization of the active site. In the presence of OAA His176 becomes hydrogen-bonded to the carbonyl O atom of OAA, whereas three arginine residues become salt-bridged to OAA carboxylate groups, namely Arg152, Arg83 and Arg89. Wireframe mesh surfaces in the top panels (gray) indicate 2F _o − F _c electron-density maps contoured at 1.5σ and the bottom panels (green) show isomorphous F _LIGAND − F _APO Fourier difference maps around NAD⁺ (contoured at −3.0σ) and OAA/APR (contoured at −2.0σ). Note that a water molecule ‘w’ (red) in the apo MexMDH structure is located in a similar position to that of the OAA carboxylate coordinating Arg152, leading to a discontinuity in the corresponding isomorphous difference map. Mechanistically relevant hydrogen-bond interactions are depicted as dashed lines.

Figure 3

Cartoon representation of secondary-structure matching superposition of the structures of MexMDH in the NAD⁺-bound (blue) and OAA/APR-bound (orange) forms. The regions experiencing significant conformational changes upon OAA binding are highlighted in green, namely the α-­helical segment SRDDLIG (residues 88–94) in α3, which acts as the active-site door, and the GGHG loop (residues 174–177), which harbors the essential residue His176.

Figure 4

Secondary-structure matching superposition of NAD⁺-bound (blue) and OAA-bound (orange) MexMDH structures, suggesting the likely binding mode of OAA in the presence of NADH. Dashed lines indicate catalytically important hydrogen-bond interactions following the same color scheme, except for the red dashed line, which indicates the distance of 2 Å between the OAA carbonyl O atom and C4 of the nicotinamide ring holding the reactive hydride.