Pyrococcus furiosus is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 105؇C by fermenting carbohydrates and peptides. Cell extracts have been previously shown to contain an unusual acetyl coenzyme A (acetyl-CoA) synthetase (ACS) which catalyzes the formation of acetate and ATP from acetyl-CoA by using ADP and phosphate rather than AMP and PP i . We show here that P. furiosus contains two distinct isoenzymes of ACS, and both have been purified.

Cell extracts of the proteolytic and hyperthermophilic archaea Thermococcus litoralis, Thermococcus sp. strain ES-1, Pyrococcus furiosus, and Pyrococcus sp. strain ES-4 contain an enzyme which catalyzes the coenzyme A-dependent oxidation of branched-chain 2-ketoacids coupled to the reduction of viologen dyes or ferredoxin. This enzyme, termed VOR (for keto-valine-ferredoxin oxidoreductase), has been purified from all four organisms. All four VORs comprise four different subunits and show amino-terminal sequence homology. T. litoralis VOR has an M r of ca. 230,000, with subunit M r values of 47,000 (␣), 34,000 (␤), 23,000 (␥), and 13,000 (␦). It contains about 11 iron and 12 acid-labile sulfide atoms and 13 cysteine residues per heterotetramer (␣␤␥␦), but thiamine pyrophosphate, which is required for catalytic activity, was lost during purification. The most efficient substrates (k cat /K m > 1.0 M ؊1 s ؊1; K m < 100 M) for the enzyme were the 2-ketoacid derivatives of valine, leucine, isoleucine, and methionine, while pyruvate and aryl pyruvates were very poor substrates (k cat /K m < 0.2 M ؊1 s ؊1) and 2-ketoglutarate was not utilized. T. litoralis VOR also functioned as a 2-ketoisovalerate synthase at 85؇C, producing 2-ketoisovalerate and coenzyme A from isobutyryl-coenzyme A (apparent K m , 250 M) and CO 2 (apparent K m , 48 mM) with reduced viologen as the electron donor. The rate of 2-ketoisovalerate synthesis was about 5% of the rate of 2-ketoisovalerate oxidation. The optimum pH for both reactions was 7.0. A mechanism for 2-ketoisovalerate oxidation based on data from substrate-induced electron paramagnetic resonance spectra is proposed, and the physiological role of VOR is discussed.In the last decade, a remarkable group of microorganisms capable of growing at temperatures around 100ЊC have been isolated from geothermally heated habitats (59,60). Virtually all of these so-called hyperthermophiles are classified as archaea (formerly archaebacteria [68]). Most of them are obligately anaerobic organisms and obtain energy for growth by either fermentation, sulfate reduction, or methanogenesis (1, 2, 28). The fermentative hyperthermophiles typically require elemental sulfur (S 0 ), which is reduced to H 2 S, for growth, although some, such as species of Pyrococcus and Thermococcus, grow well in the absence of S 0 (22,38,48). A characteristic of these organisms is their ability to ferment peptides, and some can also utilize carbohydrates (28,59,60). The pathways of carbohydrate fermentation have recently been shown to resemble the well-known bacterial pathways but with some unique additions and possibly a combination of different pathways (29,46,56).Only limited information is available on the degradation of amino acids by hyperthermophilic archaea (2, 28). Several of these organisms have been shown to contain high protease (9, 21, 33), glutamate dehydrogenase (16,19,40,54) and aromatic amino acid transaminase (3, 4) activities, and these enzymes have been purified from one or more species. In addition, three ...

Thermococcus litoralis is a strictly anaerobic archaeon (archaebacterium) that grows at temperatures up to 98؇C by fermenting peptides. It is known to contain three distinct ferredoxin-dependent, 2-keto acid oxidoreductases, which use pyruvate, aromatic 2-keto acids such as indolepyruvate, or branched-chain 2-keto acids such as 2-ketoisovalerate, as their primary substrates. We show here that T. litoralis also contains a fourth member of this family of enzymes, 2-ketoglutarate ferredoxin oxidoreductase (KGOR). In the presence of coenzyme A, KGOR catalyzes the oxidative decarboxylation of 2-ketoglutarate to succinyl coenzyme A and CO 2 and reduces T. litoralis ferredoxin. The enzyme was oxygen sensitive (half-life of ϳ5 min) and was purified under anaerobic conditions. It had an M r of approximately 210,000 and appeared to be an octomeric enzyme (␣ 2 ␤ 2 ␥ 2 ␦ 2 ) with four different subunits with M r s of 43,000 (␣), 29,000 (␤), 23,000 (␥), and 10,000 (␦). The enzyme contained 0.9 mol of thiamine PP i and at least four [4Fe-4S] clusters per mol of holoenzyme as determined by metal analyses and electron paramagnetic resonance spectroscopy. Significant amounts of other metals (Cu, Zn, Mo, W, and Ni) were not present (<0.1 mol/mol of holoenzyme). Pure KGOR did not utilize other 2-keto acids, such as pyruvate, indolepyruvate, or 2-ketoisovalerate, as substrates, and the apparent K m values for 2-ketoglutarate, coenzyme A, T. litoralis ferredoxin, and thiamine PP i were approximately 250, 40, 8, and 9 M, respectively. The enzyme was virtually inactive at 25؇C and exhibited optimal activity above 90؇C (at pH 8.0) and at pH 8.0 (at 80؇C). KGOR was quite thermostable, with a half-life at 80؇C (under anaerobic conditions) of about 2 days. An enzyme analogous to KGOR has been previously purified from a mesophilic archaeon, but the molecular properties of T. litoralis KGOR more closely resemble those of the other oxidoreductases from hyperthermophiles. In contrast to these enzymes, however, KGOR appears to have a biosynthetic function rather than a role in energy conservation.The oxidative decarboxylation of 2-keto acids such as pyruvate and 2-ketoglutarate to their acyl coenzyme A (acyl-CoA) derivatives is a key reaction of intermediary metabolism in virtually all organisms and is catalyzed by two types of thiamine PP i (TPP)-containing enzymes. Most aerobic organisms contain large, multienzyme complexes which function as 2-keto acid dehydrogenases. These contain lipoic acid and flavin as prosthetic groups, use NAD as an electron acceptor, and have been extensively studied (43). In contrast, anaerobic organisms typically utilize 2-keto acid oxidoreductases to carry out the same reaction. Unlike the dehydrogenases, these enzymes contain iron-sulfur (FeS) clusters, lack both flavin and lipoic acid, and use redox proteins such as ferredoxin or flavodoxin as electron acceptors.Pyruvate oxidoreductases (PORs) have been purified from a variety of microorganisms. They can be classified into three groups based on their subunit co...

Yeast promoter regions are often more accessible to nuclear proteins than are nonpromoter regions. As assayed by HinfI endonuclease cleavage in living yeast cells, HinfI sites located in the promoters of all seven genes tested were 5-to 20-fold more accessible than sites in adjacent nonpromoter regions. HinfI hypersensitivity within the his3 promoter region is locally determined, since it was observed when this region was translocated to the middle of the ade2 structural gene. Detailed analysis of the his3 promoter indicated that preferential accessibility is not determined by specific elements such as the Gcn4 binding site, poly(dA-dT) sequences, TATA elements, or initiator elements or by transcriptional activity. However, progressive deletion of the promoter region in either direction resulted in a progressive loss of HinfI accessibility. Preferential accessibility is independent of the Swi-Snf chromatin remodeling complex, Gcn5 histone acetylase complexes Ada and SAGA, and Rad6, which ubiquitinates histone H2B. These results suggest that preferential accessibility of the his3 (and presumably other) promoter regions is determined by a general property of the DNA sequence (e.g., base composition or a related feature) rather than by defined sequence elements. The organization of the compact yeast genome into inherently distinct promoter and nonpromoter regions may ensure that transcription factors bind preferentially to appropriate sites in promoters rather than to the excess of irrelevant but equally high-affinity sites in nonpromoter regions.

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