Crystal structure of butyrate kinase 2 from Thermotoga maritima, a member of the ASKHA superfamily of phosphotransferases

Abstract

The enzymatic transfer of phosphoryl groups is central to the control of many cellular processes. One of the phosphoryl transfer mechanisms, that of acetate kinase, is not completely understood. Besides better understanding of the mechanism of acetate kinase, knowledge of the structure of butyrate kinase 2 (Buk2) will aid in the interpretation of active-site structure and provide information on the structural basis of substrate specificity. The gene buk2 from Thermotoga maritima encodes a member of the ASKHA (acetate and sugar kinases/heat shock cognate/actin) superfamily of phosphotransferases. The encoded protein Buk2 catalyzes the phosphorylation of butyrate and isobutyrate. We have determined the 2.5-A crystal structure of Buk2 complexed with (beta,gamma-methylene) adenosine 5'-triphosphate. Buk2 folds like an open-shelled clam, with each of the two domains representing one of the two shells. In the open active-site cleft between the N- and C-terminal domains, the active-site residues consist of two histidines, two arginines, and a cluster of hydrophobic residues. The ATP binding region of Buk2 in the C-terminal domain consists of abundant glycines for nucleotide binding, and the ATP binding motif is similar to those of other members of the ASKHA superfamily. The enzyme exists as an octamer, in which four disulfide bonds form between intermolecular cysteines. Sequence alignment and structure superposition identify the simplicity of the monomeric Buk2 structure, a probable substrate binding site, the key residues in catalyzing phosphoryl transfer, and the substrate specificity differences among Buk2, acetate, and propionate kinases. The possible enzyme mechanisms are discussed.

FIG. 1.

Overview of the final 2.5-Å 2fo-fc electron density map contoured at 1.0 σ level and fitted with the final model around residue Val 180.

FIG. 2.

Ribbon representation of the structure of Buk2. The blue color of the N terminus changes gradually to the red color of the C terminus. The N- and C-terminal domains are labeled N and C, respectively. AMPPCP is rendered in ball-and-stick mode, and the formate molecule is rendered in CPK mode.

FIG. 3.

Topology diagram of Buk2. The secondary structures conserved in the ASKHA superfamily are rendered gray, and the inserts are shown in white. Rectangles indicate helices; arrows indicate β strands.

FIG. 4.

Stereo view of the active site of Buk2. Selected residues are shown as balls and sticks, as are formate and AMPPCP, and are colored according to atom type, where oxygen atoms are red, nitrogen atoms are blue, phosphorus atoms are purple, and carbon atoms are black.

FIG. 5.

Stereo view of the AMPPCP binding region. The four elements of secondary structure around AMPPCP are helix αe (Asp 255 to Gln 265), helix α2A′ (Gly 303 to Ala 306), a β turn (Mse 183 to Ile 187), and a U-shaped turn (Thr 211 to Arg 214). AMPPCP is rendered in ball-and-stick mode.

FIG. 6.

(a) AMPPCP and 2fo-fc map contoured at 1.0 σ level. (b) Schematic diagram of AMPPCP binding to Buk2. Shown are all the residues hydrogen bonded to AMPPCP within 3.5 Å.

FIG. 7.

Stereo view of the probable isobutyrate binding site. The C-terminal domains of Buk2 and glycerol kinase are superimposed. Shown are the residues of Buk2 within a cover radius of 8 Å from glycerol. Only glycerol (red), phosphate (orange), and ADP (blue) from the glycerol kinase structure are included in the representation.

FIG. 8.

Octamer of Buk2. Each monomer is rendered in a different color, and AMPPCP is represented in ball-and-stick mode. The octamer can be viewed as being composed of four dimers, and the dimer is similar to that of acetate kinase. For example, the green and red monomers form a bird-like dimer. The eight cysteines in the octamer and the corresponding four disulfide bonds are represented in CPK mode.

FIG. 9.

Superposition of the Cα atoms of Buk2, acetate kinase (monomer A, PDB code 1G99), and propionate kinase (PDB code 1X3M). Buk2 is in green, acetate kinase is in both blue and red, and propionate kinase is in both cyan and purple. The N- and C-terminal domains are labeled N and C, respectively. AMPPCP is represented in ball-and-stick mode. The N-terminal β sheets from acetate and propionate kinases are not well superposed, as there are different relative rotations between the N- and C-terminal domains of acetate and propionate kinases.