Structure of D-lactate dehydrogenase from Aquifex aeolicus complexed with NAD(+) and lactic acid (or pyruvate)

Abstract

The crystal structure of D-lactate dehydrogenase from Aquifex aeolicus (aq_727) was determined to 2.12 A resolution in space group P2(1)2(1)2(1), with unit-cell parameters a = 90.94, b = 94.43, c = 188.85 A. The structure was solved by molecular replacement using the coenzyme-binding domain of Lactobacillus helveticus D-lactate dehydrogenase and contained two homodimers in the asymmetric unit. Each subunit of the homodimer was found to be in a ;closed' conformation with the NADH cofactor bound to the coenzyme-binding domain and with a lactate (or pyruvate) molecule bound at the interdomain active-site cleft.

Figure 1

(a) Acrylamide SDS–PAGE analysis of d-lactate dehydrogenase from A. aeolicus overproduced in E. coli. (b) Crystals of A. aeolicus aq_727 prior to flash-cooling in glycerol cryoprotectant.

Figure 2

Functional homodimers of three members of the d-lactate dehydrogenase family. (a) A. aeolicus d-LDH is in the closed conformation, with a pyruvate/lactate ligand (black) bound at the NAD⁺-binding site (magenta) in both subunits of the dimer. (b) The asymmetric structure from L. bulgaricus (PDB code 1j49; Razeto et al., 2002 ▶) is shown, with a sulfate ion (black) in place of the substrate in one partially closed subunit and with the second (apo) subunit in the open conformation. (c) The fully open apo structure from L. helveticus (PDB code 2dld). The coenzyme-binding domain of 2dld was used as the molecular-replacement search model. (d) Superposition of the coenzyme-binding domains of A. aeolicus (green) and L. helveticus (brown) monomers, shown as ribbons in two perpendicular orientations with the NAD⁺ cofactor omitted for clarity. The figure illustrates the following: (i) the rotation of the catalytic domain about the ‘hinge’ axis, (ii) the locations of the residues involved in hydrogen bonding in the closed conformation, with residues from the catalytic and coenzyme-binding domains shown as yellow and black sticks, respectively, and (iii) the B-factor distribution in the monomer, with larger thermal disorder of the catalytic domain indicated by increased thickness of the ribbons. The r.m.s. deviations of the superposition of the coenzyme-binding domains of L. bulgaricus and L. helveticus with A. aeolicus are 1.0 and 0.93 Å, respectively.

Figure 3

(a) The active site of subunit A of A. aeolicus d-LDH (subunits B, C and D in the asymmetric unit were identical). (a) The orientation of the substrate bound in the cleft between the two domains, with the intermolecular contacts to the protein residues (length 2.7–2.9 Å) indicated by dashed lines. The catalytic domain is distinguished from the coenzyme-binding domain by having its C atoms coloured yellow. (b) The 2F _o − F _c electron-density map at the active site, contoured at the 1.2σ level. The electron density on the coenzyme has been omitted for clarity. (c) The F _o − F _c OMIT map at the active site showing electron density at the 3σ level with the substrate omitted from the model. (d) The quality of the 2F _o − F _c electron-density map for the NAD⁺ cofactor contoured at 1.2σ. The molecular-graphics figures were all obtained using PyMOL (DeLano, 2008 ▶).