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

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.

A continuous assay of acetate kinase activity: measurement of inorganic phosphate release generated by hydroxylaminolysis of acetyl phosphate.

Acetate kinase, a member of the ASKHA (Acetate and Sugar Kinases, Hsp70, Actin) phosphotransferase superfamily is a central enzyme in prokaryotic carbon and energy metabolism. Recently extensive structural and biochemical studies of acetate kinase and related carboxylate kinases have been conducted. Analysis of the kinetic properties of wild-type and mutant enzymes has been impeded by the nature of the current assays for acetate kinase activity. These assays have the disadvantages of being either discontinuous or insensitive or of utilizing compounds that interfere with activity measurements. We have developed a novel continuous assay that depends on the purine nucleoside phosphorylase-based spectroscopic measurement of the inorganic phosphate generated by hydroxylaminolysis of one of the products of the acetate kinase reaction, acetyl phosphate. This assay has enabled a determination of the kinetic parameters of the Thermotoga maritima acetate kinase that indicates a lower K(m) for acetate than previously published.

Origin of exopolyphosphatase processivity: Fusion of an ASKHA phosphotransferase and a cyclic nucleotide phosphodiesterase homolog.

The Escherichia coli Ppx protein is an exopolyphosphatase that degrades long-chain polyphosphates in a highly processive reaction. It also hydrolyzes the terminal 5' phosphate of the modified nucleotide guanosine 5' triphosphate 3' diphosphate (pppGpp). The structure of Ppx has been determined to 1.9 A resolution by X-ray crystallography. The exopolyphosphatase is an ASKHA (acetate and sugar kinases, Hsp70, actin) phosphotransferase with an active site found in a cleft between the two amino-terminal domains. Analysis of the active site indicates that among the ASKHA phosphotranferases of known structure, Ppx is the closest to the ectonucleoside triphosphate diphosphohydrolases. A third domain forms a six-helix claw that is similar to the catalytic core of the eukaryotic cyclic nucleotide phosphodiesterases. Most of the 29 sulfate ions bound to the Ppx dimer occupy sites where the polyP chain likely binds. An aqueduct that passes through the enzyme provides a physical basis for the enzyme's high processivity.

Crystallization of butyrate kinase 2 from Thermotoga maritima mediated by vapor diffusion of acetic acid.

The sitting-drop method of crystallization uses the evaporation of water to increase the concentration of the protein and precipitant in the drop. The presence of other volatile components, such as acetic acid, can have a marked impact on crystallization. A member of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of proteins, isobutyrate kinase (Buk2) from Thermotoga maritima, was expressed in Escherichia coli with six histidine residues added to the C-terminus. The purified protein was crystallized in a sitting drop with a well solution consisting of 1.7-3.0 M sodium formate, with the pH of the well solution alone adjusted to 4.5 with acetic acid. Diffraction data collected at 100 K show that the crystals diffract to 3.1 A and belong to space group I422, with unit-cell parameters a = b = 198.12, c = 58.93 A. Both the crystal form and the results of dynamic light-scattering studies suggest that Buk2 is an octomer, the first to be identified in the ASKHA superfamily.

Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme, benzoylformate decarboxylase.

Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.