Urkinase: structure of acetate kinase, a member of the ASKHA superfamily of phosphotransferases

Abstract

Acetate kinase, an enzyme widely distributed in the Bacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the alpha-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.

FIG. 1

Structure of acetate kinase. The structure of the acetate kinase dimer (A) and a view with a 90° rotation around a horizontal axis (B) are shown. The two monomers of the dimer are shown in green and blue. The C-terminal domains, at the center, form the dimer interface. The ADP and sulfate molecules in the active site (between the N and C domains) are shown in space-filling models. The structure contains 801 of the 816 residues in the dimer, with the missing residues located at solvent-exposed regions following the C-terminal helix. (C) Stereoview of monomer A of acetate kinase, numbered every 20 residues.

FIG. 2

Topology diagram of acetate kinase. Secondary structures conserved in the ASKHA family (the duplicated βββαβαβα core) are rendered gray, and the inserts are shown in white. In standard nomenclature, the βββαβαβα subdomains are denoted IA (left) and IIA (right). In acetate kinase, the core β strands in domain IA are numbered 1 to 5, whereas those in domain IIA are numbered 1′ to 5′.

FIG. 3

Stereoview of the superposition of the C-terminal domains of acetate kinase and of Hsc 70. The conserved ASHKA core is colored yellow for acetate kinase (with the remainder dark gray) and red for Hsc 70 (with the remainder white). The graphics program O was used to calculate the superposition matrix. The terminal helix, which extends into the N-terminal domain, is not shown.

FIG. 4

Stereoviews of the active site of acetate kinase. Conserved ASKHA secondary structures are pink and purple, and inserted secondary-structural elements are gray. Loops in the N-terminal domain are green, and those in the C-terminal domain are blue. (A) The binding site. It is likely that the sulfate occupies the position where the phosphate of acetyl phosphate would bind. No magnesium ion is apparent in our current structure despite the inclusion of 750 μM MgCl₂ in the crystallization conditions. (B) The proposed site of acetate binding. VOIDOO (27) was used to locate solvent-accessible cavities. The cavity shown could easily accommodate the methyl group of acetate or acetyl phosphate, positioning the phosphate roughly where the sulfate is located. As shown, the center of the cavity is 4.2 Å away from the sulfate.