RESUMEN

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.

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The role of histidine in the catalytic mechanism of acetate kinase from Methanosarcina thermophila was investigated by diethylpyrocarbonate inactivation and site-directed mutagenesis. Inactivation was accompanied by an increase in absorbance at 240 nm with no change in absorbance at 280 nm, and treatment of the inactivated enzyme with hydroxylamine restored 95% activity, results that indicated diethylpyrocarbonate inactivates the enzyme by the specific modification of histidine. The substrates ATP, ADP, acetate, and acetyl phosphate protected against inactivation suggesting at least one active site where histidine is modified. Correlation of residual activity with the number of histidines modified, as determined by absorbance at 240 nm, indicated that a maximum of three histidines are modified per subunit, two of which are essential for full inactivation. Comparison of the M. thermophila acetate kinase sequence with 56 putative acetate kinase sequences revealed eight highly conserved histidines, three of which (His-123, His-180, and His-208) are perfectly conserved. Diethylpyrocarbonate inactivation of the eight histidine --> alanine variants indicated that His-180 and His-123 are in the active site and that the modification of both is necessary for full inactivation. Kinetic analyses of the eight variants showed that no other histidines are important for activity. Analysis of additional His-180 variants indicated that phosphorylation of His-180 is not essential for catalysis. Possible functions of His-180 are discussed.

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Site-directed mutagenesis is a powerful tool for identifying active-site residues essential for catalysis; however, this approach has only recently become available for acetate kinase. The enzyme from Methanosarcina thermophila has been cloned and hyper-produced in a highly active form in Escherichia coli (recombinant wild-type). The role of arginines in this acetate kinase was investigated. Five arginines (R91, R175, R241, R285, and R340) in the M. thermophila enzyme were selected for individual replacement based on their high conservation among sequences of acetate kinase homologues. Replacement of R91 or R241 with alanine or leucine produced variants with specific activities less than 0.1% of the recombinant wild-type enzyme. The circular dichroism spectra and other properties of these variants were comparable to those of recombinant wild-type, indicating no global conformational changes. These results indicate that R91 and R241 are essential for activity, consistent with roles in catalysis. The variant produced by conservative replacement of R91 with lysine had approximately 2% of recombinant wild-type activity, suggesting a positive charge is important in this position. The K(m) value for acetate of the R91K variant increased greater than 10-fold relative to recombinant wild-type, suggesting an additional role for R91 in binding this substrate. Activities of both the R91A and R241A variants were rescued 20-fold when guanidine or derivatives were added to the reaction mixture. The K(m) values for ATP of the rescued variants were similar to those of recombinant wild-type, suggesting that the rescued activities are the consequence of replacement of important functional groups and not changes in the catalytic mechanism. These results further support roles for R91 and R241 in catalysis. Replacement of R285 with alanine, leucine, or lysine had no significant effect on activity; however, the K(m) values for acetate increased 6-10-fold, suggesting R285 influences the binding of this substrate. Phenylglyoxal inhibition and substrate protection experiments with the recombinant wild-type enzyme and variants were consistent with the presence of one or more essential arginine residues in the active site as well as with roles for R91 and R241 in catalysis. It is proposed that R91 and R241 function to stabilize the previously proposed pentacoordinate transition state during direct in-line transfer of the gamma-phosphate of ATP to acetate. The kinetic characterization of variants produced by replacement of R175 and R340 with alanine, leucine, or lysine indicated that these residues are not involved in catalysis but fulfill important structural roles.

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Periplasmic cyclic beta-glucans of Rhizobium species provide important functions during plant infection and hypo-osmotic adaptation. In Sinorhizobium meliloti (also known as Rhizobium meliloti), these molecules are highly modified with phosphoglycerol and succinyl substituents. We have previously identified an S. meliloti Tn5 insertion mutant, S9, which is specifically impaired in its ability to transfer phosphoglycerol substituents to the cyclic beta-glucan backbone (M. W. Breedveld, J. A. Hadley, and K. J. Miller, J. Bacteriol. 177:6346-6351, 1995). In the present study, we have cloned, sequenced, and characterized this mutation at the molecular level. By using the Tn5 flanking sequences (amplified by inverse PCR) as a probe, an S. meliloti genomic library was screened, and two overlapping cosmid clones which functionally complement S9 were isolated. A 3.1-kb HindIII-EcoRI fragment found in both cosmids was shown to fully complement mutant S9. Furthermore, when a plasmid containing this 3.1-kb fragment was used to transform Rhizobium leguminosarum bv. trifolii TA-1JH, a strain which normally synthesizes only neutral cyclic beta-glucans, anionic glucans containing phosphoglycerol substituents were produced, consistent with the functional expression of an S. meliloti phosphoglycerol transferase gene. Sequence analysis revealed the presence of two major, overlapping open reading frames within the 3.1-kb fragment. Primer extension analysis revealed that one of these open reading frames, ORF1, was transcribed and its transcription was osmotically regulated. This novel locus of S. meliloti is designated the cgm (cyclic glucan modification) locus, and the product encoded by ORF1 is referred to as CgmB.

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Acetate kinase catalyzes the reversible phosphorylation of acetate (CH3COO- + ATP<-->CH3CO2PO3(2-) + ADP). A mechanism which involves a covalent phosphoryl-enzyme intermediate has been proposed, and chemical modification studies of the enzyme from Escherichia coli indicate an unspecified glutamate residue is phosphorylated (J. A. Todhunter and D. L. Purich, Biochem. Biophys. Res. Commun. 60:273-280, 1974). Alignment of the amino acid sequences for the acetate kinases from E. coli (Bacteria domain), Methanosarcina thermophila (Archaea domain), and four other phylogenetically divergent microbes revealed high identity which included five glutamates. These glutamates were replaced in the M. thermophila enzyme to determine if any are essential for catalysis. The histidine-tagged altered enzymes were produced in E. coli and purified to electrophoretic homogeneity by metal affinity chromatography. Replacements of E384 resulted in either undetectable or extremely low kinase activity, suggesting E384 is essential for catalysis which supports the proposed mechanism. Replacement of E385 influenced the Km values for acetate and ATP with only moderate decreases in k(cat), which suggests that this residue is involved in substrate binding but not catalysis. The unaltered acetate kinase was not inactivated by N-ethylmaleimide; however, replacement of E385 with cysteine conferred sensitivity to N-ethylmaleimide which was prevented by preincubation with acetate, acetyl phosphate, ATP, or ADP, suggesting that E385 is located near the active site. Replacement of E97 decreased the Km value for acetate but not ATP, suggesting this residue is involved in binding acetate. Replacement of either E32 or E334 had no significant effects on the kinetic constants, which indicates that neither residue is essential for catalysis or significantly influences the binding of acetate or ATP.

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The cyclic beta-(1,2)-glucans of Rhizobium meliloti and Agrobacterium tumefaciens play an important role during hypoosmotic adaptation, and the synthesis of these compounds is osmoregulated. Glucosyltransferase, the enzyme responsible for cyclic beta-(1,2)-glucan biosynthesis, is present constitutively, suggesting that osmotic regulation of the biosynthesis of these glucans occurs through modulation of enzyme activity. In this study, we examined regulation of cyclic glucan biosynthesis in vitro with membrane preparations from R. meliloti. The results show that ionic solutes inhibit glucan synthesis, even when they are present at low concentrations (e.g., 10 mM). In contrast, neutral solutes (glucose, sucrose, and the compatible solutes glycine betaine and trehalose) were found to stimulate glucan synthesis in vitro when they were present at high concentrations (e.g., 1 M). Furthermore, high concentrations of these neutral solutes were shown to compensate for the inhibition of glucosyltransferase activity by ionic solutes. Consistent with their ionic character, the compatible solute potassium glutamate and the osmoprotectant choline chloride inhibited glucosyltransferase activity in vitro. The results suggest that intracellular ion concentrations, intracellular osmolarity, and intracellular concentrations of nonionic compatible solutes all act as important determinants of glucosyltransferase activity in vivo. Additional experiments were performed with an ndvA mutant defective for transport of cyclic glucans and an ndvB mutant that produces a C-terminal truncated glucosyltransferase. Cyclic beta-(1,2)-glucan biosynthesis, although reduced, was found to be osmoregulated in both mutants. These results reveal that NdvA and the C terminus of NdvB are not required for osmotic regulation of cyclic beta-(1,2)-glucan biosynthesis.

RESUMEN

The unique biochemical properties of acetate kinase present a classic conundrum in the study of the mechanism of enzyme-catalyzed phosphoryl transfer. Large, single crystals of acetate kinase from Methanosarcina thermophila were grown from a solution of ammonium sulfate in the presence of ATP. The crystals diffract to beyond 1.7 A resolution. Analysis of X-ray data from the crystals is consistent with a space group of C2 and unit cell dimensions a = 181 A, b = 67 A, c = 83 A, beta = 103 degrees. Diffraction data have been collected from the crystals at 110 and 277 K. Data collected at 277 K extend to lower resolution, but are more reproducible. The orientation of a noncrystallographic two-fold axis of symmetry has been determined. Based on an analysis of the predicted amino acid sequences of acetate kinase from several organisms, we hypothesize that acetate kinase is a member of the sugar kinase/actin/hsp70 structural family.

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