Cloning of a Gene Encoding Acetate Kinase from Methanosarcina mazei 2-P Isolated from a Japanese Paddy Field Soil

The ack gene encoding acetate kinase from the mesophilic Methanosarcina mazei 2-P, isolated from a paddy field soil in Japan, was cloned, sequenced, and functionally expressed in Escherichia coli. The terminal region of the putative pta gene, probably encoding phosphotransacetylase, was found upstream of the ack gene. The deduced amino acid sequence of the acetate kinase is 86.5% identical to that of the Methanosarcina thermophila acetate kinase. The activity of the His₆-tagged acetate kinase purified from E. coli JM109 was optimal at 35°C. Received: 28 January 2002 / Accepted: 27 February 2002     

Characterization of Methanosarcina mazeii TMA isolated from a paddy field soil

We isolated a methanogenic strain, designated as strain TMA (=DSM 9195), from an enrichment culture inoculated with a Japanese paddy field soil. Strain TMA was Gram positive and strictly anaerobic. Cell shape was pseudosarcina-like, and cells were nonmotile. The strain was able to use methylamines, methanol, H₂–CO₂, and acetate as substrates for methanogenesis, but did not utilize formate. The optimum temperature and optimum pH were 30–37°C and 6.5–7.5 respectively. The G+C content of the DNA was 42.1 mol %. Strain TMA had DNA-DNA hybridization values of more than 80% with Methanosarcina mazeii S-6^T (T = type strain). On the basis of phenotypic and genotypic characteristics, we identified strain TMA as M. mazeii. This is the first methylotrophic methanogen isolated from a paddy field soil and identified to the species level.     

Characterization of a feedback-resistant mevalonate kinase from the archaeon Methanosarcina mazei

The mevalonate pathway is utilized for the biosynthesis of isoprenoids in many bacterial, eukaryotic, and archaeal organisms. Based on previous reports of its feedback inhibition, mevalonate kinase (MVK) may play an important regulatory role in the biosynthesis of mevalonate pathway-derived compounds. Here we report the purification, kinetic characterization, and inhibition analysis of the MVK from the archaeon Methanosarcina mazei. The inhibition of the M. mazei MVK by the following metabolites derived from the mevalonate pathway was explored: dimethylallyl diphosphate (DMAPP), geranyl pyrophosphate (GPP), farnesyl pyrophosphate (FPP), isopentenyl monophosphate (IP), and diphosphomevalonate. M. mazei MVK was not inhibited by DMAPP, GPP, FPP, diphosphomevalonate, or IP, a proposed intermediate in an alternative isoprenoid pathway present in archaea. Our findings suggest that the M. mazei MVK represents a distinct class of mevalonate kinases that can be differentiated from previously characterized MVKs based on its inhibition profile.     

Cloning, sequence analysis, and hyperexpression of the genes encoding phosphotransacetylase and acetate kinase from Methanosarcina thermophila.

The genes for the acetate-activating enzymes, acetate kinase and phosphotransacetylase (ack and pta), from Methanosarcina thermophila TM-1 were cloned and sequenced. Both genes are present in only one copy per genome, with the pta gene adjacent to and upstream of the ack gene. Consensus archaeal promoter sequences are found upstream of the pta coding region. The pta and ack genes encode predicted polypeptides with molecular masses of 35,198 and 44,482 Da, respectively. A hydropathy plot of the deduced phosphotransacetylase sequence indicates that it is a hydrophobic polypeptides; however, no membrane-spanning domains are evident. Comparison of the amino acid sequences deduced from the M. thermophila and Escherichia coli ack genes indicate similar subunit molecular weights and 44% identity (60% similarity). The comparison also revealed the presence of several conserved arginine, cysteine, and glutamic acid residues. Arginine, cysteine, and glutamic acid residues have previously been implicated at or near the active site of the E. coli acetate kinase. The pta and ack genes were hyperexpressed in E. coli, and the overproduced enzymes were purified to homogeneity with specific activities higher than those of the enzymes previously purified from M. thermophila. The overproduced phosphotransacetylase and acetate kinase migrated at molecular masses of 37,000 and 42,000 Da, respectively. The activity of the acetate kinase is optimal at 65 degrees C and is protected from thermal inactivation by ATP. Diethylpyrocarbonate and phenylglyoxal inhibited acetate kinase activity in a manner consistent with the presence of histidine and arginine residues at or near the active site; however, the thiol-directed reagents 5,5'-dithiobis (2-nitrobenzoic acid) and N-ethylmaleimide were ineffective.     

Bacillus huizhouensis sp. nov., isolated from a paddy field soil

A Gram-stain positive, facultative aerobic bacterium, designated as strain GSS03^T, was isolated from a paddy field soil. The cells were observed to be endospore forming, rod-shaped and motile with flagella. The organism was found to grow optimally at 35 °C at pH 7.0 and in the presence of 1 % NaCl. The strain was classified as a novel taxon within the genus Bacillus on the basis of phenotypic and phylogenetic analyses. The closest phylogenetic relatives were identified as Bacillus psychrosaccharolyticus DSM 6^T (97.61 %), Bacillus muralis DSM 16288^T (97.55 %), Bacillus asahii JCM 12112^T (97.48 %), Bacillus simplex DSM 1321^T (97.48 %) and “Bacillus frigoritolerans” DSM 8801^T (97.38 %). The menaquinone was identified as MK-7, the major cellular fatty acid was identified as anteiso-C_15:0 and the major cellular polar lipids as phosphatidylethanolamine, phosphatidylmonomethylethanolamine, diphosphatidylglycerol, phosphatidylglycerol and three unknown polar lipids. The DNA G+C content was determined to be 40.2 mol%. The DNA–DNA relatedness with the closest relatives was below 48 %. Therefore, on the basis of all the results, strain GSS03^T is considered to represent a novel species within the genus Bacillus, for which the name Bacillus huizhouensis sp. nov. is proposed. The type strain is GSS03^T (=KCTC 33172^T =CCTCC AB 2013237^T).     

Characterization of Methanosarcina mazei N2M9705 Isolated from an Aquaculture Fishpond

A new methanogenic isolate, designated as strain N2M9705 (=OCM 668), was isolated from an aquaculture fishpond near Wang-gong, Taiwan. This strain grew on trimethylamine and methanol, but it did not catabolize H₂-CO₂, acetate, or formate. The cells were stained Gram-negative, nonmotile, irregular coccus 0.6–0.8 μm in diameter. Gas vacuoles were observed and cell aggregated to form various sizes of granules. Cells grew optimally at 32°–37°C with 1% NaCl. The pH range of growth was 6.2–7.4, and higher pH inhibited the cell growth. The cells grew well in minimal medium, but growth was greatly stimulated by yeast extract and peptone. A comparison of 16S rDNA sequences of this organism phylogenetically related to Methanosarcina mazei. This is the first report of methyltrophic methanogenic isolated from an aquaculture fishpond. Received: 16 March 1999 / Accepted: 16 April 1999     

Methanogenesis from lactate by a co-culture of Clostridium formicoaceticum and Methanosarcina mazei

Summary A co-culture of Clostridium formicoaceticum and Methanosarcina mazei converted lactate to methane and carbon dioxide at mesophilic temperatures and pH values near 7.0. Lactate was first converted to acetate by the homoacetogen, and then to CH₄ and CO₂ by the methanogen, with the second reaction as the rate-limiting step. The methane yield was about 1.45 mol/mol lactate. These two organisms formed a mutualistic association and may be useful together with the homolactic bacterium Stretococcus lactis to convert lactose to methane. Offprint requests to: S. T. Yang     

Identification of essential arginines in the acetate kinase from Methanosarcina thermophila

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.     

The role of histidines in the acetate kinase from Methanosarcina thermophila

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.     

Cloning and characterization of a gene encoding phosphoketolase in a Lactobacillus paraplantarum isolated from Kimchi

A gene coding for phosphoketolase, a key enzyme of carbohydrate catabolism in heterofermentative lactic acid bacteria (LAB), was cloned from a Lactobacillus paraplantarum C7 and expressed in Escherichia coli. The gene is 2502 bp long and codes for a 788-amino-acids polypeptide with a molecular mass of 88.7 kDa. A Shine-Dalgamo sequence (aaggag) and an inverted-repeat terminator sequence are located upstream and downstream of the phosphoketolase gene, respectively. The gene exhibits an identity of >52% with phosphoketolases of other LAB. The phosphoketolase of Lb. paraplantarum C7 (LBPK) contains several highly conserved phosphoketolase signature regions and typical thiamine pyrophosphate (TPP) binding sites, as reported for other TPP-dependent enzymes. The phosphoketolase gene was fused to a glutathione S-transferase (GST::LBPK) gene for purification. The GST::LBPK fusion protein was detected in the soluble fraction of a recombinant Escherichia coli BL21. The GST::LBPK fusion protein was purified with a yield of 4.32 mg/400 ml by GSTrap HP affinity column chromatography and analyzed by N-terminal sequencing. LBPK was obtained by factor Xa treatment of fusion protein and the final yield was 3.78 mg/400 ml. LBPK was examined for its N-terminal sequence and phosphoketolase activity. The K(M) and Vmax values for fructose-6-phosphate were 5.08 +/- 0.057 mM (mean +/- SD) and 499.21 +/- 4.33 micromol/min/mg, respectively, and the optimum temperature and pH for the production of acetyl phosphate were 45 degrees C and 7.0, respectively.     

Crystallization of acetate kinase from Methanosarcina thermophila and prediction of its fold.

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.     

The amber codon in the gene encoding the monomethylamine methyltransferase isolated from Methanosarcina barkeri is translated as a sense codon.

Each of the genes encoding the methyltransferases initiating methanogenesis from trimethylamine, dimethylamine, or monomethylamine by various Methanosarcina species possesses one naturally occurring in-frame amber codon that does not appear to act as a translation stop during synthesis of the biochemically characterized methyltransferase. To investigate the means by which suppression of the amber codon within these genes occurs, MtmB, a methyltransferase initiating metabolism of monomethylamine, was examined. The C-terminal sequence of MtmB indicated that synthesis of this mtmB1 gene product did not cease at the internal amber codon, but at the following ochre codon. Antibody raised against MtmB revealed that Escherichia coli transformed with mtmB1 produced the amber termination product. The same antibody detected primarily a 50-kDa protein in Methanosarcina barkeri, which is the mass predicted for the amber readthrough product of the mtmB1 gene. Sequencing of peptide fragments from MtmB by Edman degradation and mass spectrometry revealed no change in the reading frame during mtmB1 expression. The amber codon position corresponded to a lysyl residue using either sequencing technique. The amber codon is thus read through during translation at apparently high efficiency and corresponds to lysine in tryptic fragments of MtmB even though canonical lysine codon usage is encountered in other Methanosarcina genes.     

Evidence for a transition state analog, MgADP-aluminum fluoride-acetate, in acetate kinase from Methanosarcina thermophila

Aluminum fluoride has become an important tool for investigating the mechanism of phosphoryl transfer, an essential reaction that controls a host of vital cell functions. Planar AlF(3) or AlF(4)(-) molecules are proposed to mimic the phosphoryl group in the catalytic transition state. Acetate kinase catalyzes phosphoryl transfer of the ATP gamma-phosphate to acetate. Here we describe the inhibition of acetate kinase from Methanosarcina thermophila by preincubation with MgCl(2), ADP, AlCl(3), NaF, and acetate. Preincubation with butyrate in place of acetate did not significantly inhibit the enzyme. Several NTPs can substitute for ATP in the reaction, and the corresponding NDPs, in conjunction with MgCl(2), AlCl(3), NaF, and acetate, inhibit acetate kinase activity. Fluorescence quenching experiments indicated an increase in binding affinity of acetate kinase for MgADP in the presence of AlCl(3), NaF, and acetate. These and other characteristics of the inhibition indicate that the transition state analog, MgADP-aluminum fluoride-acetate, forms an abortive complex in the active site. The protection from inhibition by a non-hydrolyzable ATP analog or acetylphosphate, in conjunction with the strict dependence of inhibition on the presence of both ADP and acetate, supports a direct in-line mechanism for acetate kinase.     

Site-directed mutational analysis of active site residues in the acetate kinase from Methanosarcina thermophila

Acetate kinase catalyzes the magnesium-dependent transfer of the gamma-phosphate of ATP to acetate. The recently determined crystal structure of the Methanosarcina thermophila enzyme identifies it as a member of the sugar kinase/Hsc70/actin superfamily based on the fold and the presence of five putative nucleotide and metal binding motifs that characterize the superfamily. Residues from four of these motifs in M. thermophila acetate kinase were selected for site-directed replacement and analysis of the variants. Replacement of Asp(148) and Asn(7) resulted in variants with catalytic efficiencies less than 1% of that of the wild-type enzyme, indicating that these residues are essential for activity. Glu(384) was also found to be essential for catalysis. A 30-fold increase in the magnesium concentration required for half-maximal activity of the E384A variant relative to that of the wild type implicated Glu(384) in magnesium binding. The kinetic analysis of variants and structural data is consistent with nonessential roles for active site residues Ser(10), Ser(12), and Lys(14) in catalysis. The results are discussed with respect to the acetate kinase catalytic mechanism and the relationship to other sugar kinase/Hsc70/actin superfamily members.     

Cloning of the genomic sequence encoding a processed adenylate kinase 2 pseudogene

A chromosomal DNA sequence harboring a processed AK2B pseudogene was isolated from a human genomic library. It was a variant of the AK2B gene sequence including several point mutations, deletions, and insertions. The nucleotide sequence of the ORF of the AK2B pseudogene predicted a truncated form of the AK2B mutant suggesting that the processed pseudogene is nonfunctional. A repetitive sequence, AAAAGAGAG, found in the 5' and 3' flanking regions of the pseudogene and the poly(A) tract in the 3' end junction suggest that a mRNA of AK2B may have been converted to the processed pseudogene by retrotransposition events. Previously, it was suggested that an adenylate kinase (AK) 2 related gene on chromosome 2, confirmed by Southern analysis using somatic cell hybrid cell lines, may be a processed pseudogene. It is proposed that the processed pseudogene isolated in this study may be the AK2 related nonfunctional gene localized on human chromosomes 2.