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 polypeptide; 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°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.The metabolism of acetate, whether through the production or consumption of the principal intermediate acetyl coenzyme A (acetyl-CoA), is of major importance in the physiology of both procaryotes (Bacteria and Archaea domains) and eucaryotes (Eucarya domain). Acetate is an end product in the energy-yielding metabolism of most facultatively and strictly anaerobic microbes. Phosphotransacetylase (EC 2.3.1.8) and acetate kinase (EC 2.7.2.1) are synthesized by these anaerobes to catalyze the conversion of acetyl-CoA and ADP to acetate and ATP,

Members of theTranscription factors of the Rel/NF-B family are present in most or all mammalian and avian cells and influence the expression of many genes (for a review, see references 1 and 19). These factors are related to each other over a region of about 300 amino acids called the Rel Homology Domain (RHD), which governs DNA binding and dimerization. Although each protein has a nuclear localization signal (NLS), the various homo-and heterodimers are cytoplasmic by virtue of an inhibitor protein whose binding masks the NLSs. Several different inhibitors (called IBs) are known, and they constitute a family of related proteins. Each IB contains five or six so-called ankyrin repeats, motifs of about 33 amino acids which are abundantly represented in the protein ankyrin. In ankyrin, these repeats apparently function in groups of about six (18) and constitute a protein-protein interaction domain. In addition, the inhibitors contain a C-terminal region which tends to be highly acidic and to resemble PEST sequences associated with rapid protein turnover (22). The IB N-terminal region contains two serine residues which, upon phosphorylation, trigger ubiquitination and proteasomal degradation of the inhibitor, thus freeing the dimer to enter the nucleus (4,5,7,(26)(27)(28)(29).While several crystal structures of Rel family members are known (6, 6a, 10, 20), none is known for the IB family. Nevertheless, studies with various mutants of the Rel family and/or IB proteins have led to some general conclusions about their interaction. First, a Rel family dimer binds a single IB molecule (8,12,14). Second, at least some of the contacts with IB occur in the dimerization domains of the Rel proteins. Based on the crystal structure, each monomer of p50 and RelA consists of two separate domains connected by a short hinge region. The N-terminal domain contains some (but not all) of the residues that contact DNA, while the C-terminal domain contains the remaining DNA contacts and governs dimerization. Partial deletion of the N-terminal domain of RelA did not abolish binding of IB␣ (9). Similar results were obtained with Cactus and N-terminal deletion mutants of Dorsal (Drosophila relatives of IB and Rel proteins, respectively) (11, 25). In addition, mutation of two residues in the dimerization domain of Dorsal prevented interaction with Cactus but did not affect DNA binding or dimerization (16). These studies suggest contact between IB and the C-terminal part of the RHD. The third general conclusion is that the dimer NLSs, which were not resolved in the crystal structures but are immediately downstream of the dimerization domain in the primary structure, are somehow involved in binding to IB. While in at least some cases an intact NLS may not be required for binding to IB (11,23), several studies have shown that altering the NLS can abolish binding (2,9,15,25). Involvement of the NLS would correlate nicely with the fact that the dimer NLSs are masked in a dimer-IB complex. The final generalization is that most of the IB contacts oc...

A gene (isf) encoding an iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila was cloned and sequenced. The gene was located directly upstream of the genes (pta and ack) encoding phosphotransacetylase and acetate kinase and is transcribed in the opposite direction. The amino acid sequence deduced from isf contained a cluster of cysteine residues reminiscent of proteins that accommodate either a [4Fe-4S] or [3Fe-4S] center. The protein was heterologously produced in Escherichia coli and purified to apparent homogeneity. The 29-kDa subunit molecular mass of heterologously produced Isf (determined by SDS-polyacrylamide gel electrophoresis) corresponded to the molecular mass of 30,451 Da calculated from the amino acid composition deduced from isf. Gel filtration estimated a molecular mass of 65 kDa for the native Isf indicating an alpha2 homodimer. The UV-visible absorption spectrum was characteristic of iron-sulfur flavoproteins with maxima at 484, 452, 430, 378, and 280 nm. Analyses identified 2 FMN, 7-8 non-heme iron atoms, and 6-7 acid-labile sulfur atoms per alpha2 homodimer. Comparisons of the deduced Isf sequence with sequences in available protein data bases suggested Isf is a novel iron-sulfur flavoprotein. Western blot analysis indicated the presence of Isf in extracts of acetate-grown M. thermophila. Ferredoxin stimulated the CO-dependant reduction of Isf by the CO dehydrogenase middle dotacetyl-CoA synthase complex that suggested ferredoxin is a physiological electron donor to Isf.