Pyruvate decarboxylase: a key enzyme for the oxidative metabolism of lactic acid by Acetobacter pasteurianus.

Acetobacter pasteurianus, an obligately oxidative bacterium, is the first organism shown to utilize pyruvate decarboxylase (PDC) as a central enzyme for oxidative metabolism. In plants, yeast, and other bacteria, PDC functions solely as part of the fermentative ethanol pathway. During the growth of A. pasteurianus on lactic acid, the central intermediate pyruvate is cleaved to acetaldehyde and CO(2) by PDC. Acetaldehyde is subsequently oxidized to its final product, acetic acid. The presence of the PDC enzyme in A. pasteurianus was confirmed by zymograms stained for acetaldehyde production, enzyme assays using alcohol dehydrogenase as the coupling enzyme, and by cloning and characterization of the pdc operon. A. pasteurianus pdc was also expressed in recombinant Escherichia coli. The level of PDC activity was regulated in response to growth substrate, highest with lactic acid and absent with mannitol. The translated PDC sequence (548 amino acids) was most similar to that of Zymomonas mobilis, an obligately fermentative bacterium. A second operon ( aldA) was also found which is transcribed divergently from pdc. This operon encodes a putative aldehyde dehydrogenase (ALD2; 357 amino acids) related to class III alcohol dehydrogenases and most similar to glutathione-dependent formaldehyde dehydrogenases from alpha-Proteobacteria and Anabeana azollae.

Proteasomes in the archaea: from structure to function.

Survival of cells is critically dependent on their ability to rapidly adapt to changes in the natural environment no matter how 'extreme'the habitat. An interplay between protein folding and hydrolysis is emerging as a central mechanism for stress survival and proper cell function. In eucaryotic cells, most proteins destined for destruction are covalently modified by the ubiquitin-system and then degraded in an energy-dependent mechanism by the 26S proteasome, a multicatalytic protease. The 26S proteasome is composed of a 20S proteolytic core and 19S cap (PA700) regulator which includes six AAA+ ATPase subunits. Related AAA+ proteins and 20S proteasomes are found in the archaea and Gram positive actinomycetes. In general, 20S proteasomes form a barrel-shaped nanocompartment with narrow openings which isolate rather non-specific proteolytic active-sites to the interior of the cylinder and away from interaction with cytosolic proteins. The proteasome-associated AAA+ proteins are predicted to form ring-like structures which unfold substrate proteins for entry into the central proteolytic 20S chamber resulting in an energy-dependent and processive destruction of the protein. Detailed biochemical and biophysical analysis as well as identification of proteasomes in archaea with developed genetic tools are providing a foundation for understanding the biological role of the proteasome in these unusual organisms.

Biochemical characterization of the 20S proteasome from the methanoarchaeon Methanosarcina thermophila.

The 20S proteasome from the methanoarchaeon Methanosarcina thermophila was produced in Escherichia coli and characterized. The biochemical properties revealed novel features of the archaeal 20S proteasome. A fully active 20S proteasome could be assembled in vitro with purified native alpha ring structures and beta prosubunits independently produced in Escherichia coli, which demonstrated that accessory proteins are not essential for processing of the beta prosubunits or assembly of the 20S proteasome. A protein complex with a molecular mass intermediate to those of the alpha7 ring and the 20S proteasome was detected, suggesting that the 20S proteasome is assembled from precursor complexes. The heterologously produced M. thermophila 20S proteasome predominately catalyzed cleavage of peptide bonds carboxyl to the acidic residue Glu (postglutamyl activity) and the hydrophobic residues Phe and Tyr (chymotrypsinlike activity) in short chromogenic and fluorogenic peptides. Low-level hydrolyzing activities were also detected carboxyl to the acidic residue Asp and the basic residue Arg (trypsinlike activity). Sodium dodecyl sulfate and divalent or monovalent ions stimulated chymotrypsinlike activity and inhibited postglutamyl activity, whereas ATP stimulated postglutamyl activity but had little effect on the chymotrypsinlike activity. The results suggest that the 20S proteasome is a flexible protein which adjusts to binding of substrates. The 20S proteasome also hydrolyzed large proteins. Replacement of the nucleophilic Thr1 residue with an Ala in the beta subunit abolished all activities, which suggests that only one active site is responsible for the multisubstrate activity. Replacement of beta subunit active-site Lys33 with Arg reduced all activities, which further supports the existence of one catalytic site; however, this result also suggests a role for Lys33 in polarization of the Thr1 N, which serves to strip a proton from the active-site Thr1 Ogamma nucleophile. Replacement of Asp51 with Asn had no significant effect on trypsinlike activity, enhanced postglutamyl and trypsinlike activities, and only partially reduced lysozyme-hydrolyzing activity, which suggested that this residue is not essential for multisubstrate activity.

Analysis of the CO dehydrogenase/acetyl-coenzyme A synthase operon of Methanosarcina thermophila.

The cdhABC genes encoding the respective alpha, epsilon, and beta subunits of the five-subunit (alpha, beta, gamma, delta, and epsilon) CO dehydrogenase/acetyl-coenzyme synthase (CODH/ACS) complex from Methanosarcina thermophila were cloned and sequenced. Northern (RNA) blot analyses indicated that the cdh genes encoding the five subunits and an open reading frame (ORF1) with unknown function are cotranscribed during growth on acetate. Northern blot and primer extension analyses suggested that mRNA processing and multiple promoters may be involved in cdh transcript synthesis. The putative CdhA (alpha subunit) and CdhB (epsilon subunit) proteins each have 40% identity to CdhA and CdhB of the CODH/ACS complex from Methanosaeta soehngenii. The cdhC gene encodes the beta subunit (CdhC) of the CODH/ACS complex from M. thermophila. The N-terminal 397 amino acids of CdhC are 42% identical to the C-terminal half of the alpha subunit of CODH/ACS from the acetogenic anaerobe Clostridium thermoaceticum. Sequence analysis suggested potential structures and functions for the previously uncharacterized beta subunit from M. thermophila. The deduced protein sequence of ORF1, located between the cdhC and cdhD genes, has 29% identity to NifH2 from Methanobacterium ivanovii.

A proteasome from the methanogenic archaeon Methanosarcina thermophila.

A 645-kDa proteasome was purified from Methanosarcina thermophila which had chymotrypsin-like and peptidylglutamyl-peptide hydrolase activities and contained alpha (24-kDa) and beta (22-kDa) subunits. Processing of both subunits was suggested by comparison of N-terminal sequences with the sequences deduced from the alpha- and beta-encoding genes (psmA and psmB). Alignment of deduced sequences for the alpha and beta subunits revealed high similarity; however, the N-terminal sequence of the alpha subunit contained an additional 24 amino acids that were not present in the beta subunit. The alpha and beta subunits had high sequence identity with alpha- and beta-type subunits of proteasomes from eucaryotic organisms and the distantly related archaeon Thermoplasma acidophilum. The psmB gene was transcribed in vivo as a monocistronic message from a consensus archaeal promoter. The results suggest that proteasomes are more widespread in the Archaea than previously proposed. Southern blotting experiments suggested the presence of ubiquitin-like sequences in M. thermophila.

Genetic analysis of the modABCD (molybdate transport) operon of Escherichia coli.

DNA sequence analysis of the modABCD operon of Escherichia coli revealed the presence of four open reading frames. The first gene, modA, codes for a 257-amino-acid periplasmic binding protein enunciated by the presence of a signal peptide-like sequence. The second gene (modB) encodes a 229-amino-acid protein with a potential membrane location, while the 352-amino-acid ModC protein (modC product) contains a nucleotide-binding motif. On the basis of sequence similarities with proteins from other transport systems and molybdate transport proteins from other organisms, these three proteins are proposed to constitute the molybdate transport system. The fourth open reading frame (modD) encodes a 231-amino-acid protein of unknown function. Plasmids containing different mod genes were used to map several molybdate-suppressible chlorate-resistant mutants; interestingly, none of the 40 mutants tested had a mutation in the modD gene. About 35% of these chlorate-resistant mutants were not complemented by mod operon DNA. These mutants, designated mol, contained mutations at unknown chromosomal location(s) and produced formate hydrogenlyase activity only when cultured in molybdate-supplemented glucose-minimal medium, not in L broth. This group of mol mutants constitutes a new class of molybdate utilization mutants distinct from other known mutants in molybdate metabolism. These results show that molybdate, after transport into cells by the ModABC proteins, is metabolized (activated?) by the products of the mol gene(s).

Molybdate and regulation of mod (molybdate transport), fdhF, and hyc (formate hydrogenlyase) operons in Escherichia coli.

Escherichia coli mutants with defined mutations in specific mod genes that affect molybdate transport were isolated and analyzed for the effects of particular mutations on the regulation of the mod operon as well as the fdhF and hyc operons which code for the components of the formate hydrogenlyase (FHL) complex. phi (hyc'-'lacZ+) mod double mutants produced beta-galactosidase activity only when they were cultured in medium supplemented with molybdate. This requirement was specific for molybdate and was independent of the moa, mob, and moe gene products needed for molybdopterin guanine dinucleotide (MGD) synthesis, as well as Mog protein. The concentration of molybdate required for FHL production by mod mutants was dependent on medium composition. In low-sulfur medium, the amount of molybdate needed by mod mutants for the production of half-maximal FHL activity was increased approximately 20 times by the addition of 40 mM of sulfate, mod mutants growing in low-sulfur medium transported molybdate through the sulfate transport system, as seen by the requirement of the cysA gene product for this transport. In wild-type E. coli, the mod operon is expressed at very low levels, and a mod+ merodiploid E. coli carrying a modA-lacZ fusion produced less than 20 units of beta-galactosidase activity. This level was increased by over 175 times by a mutation in the modA, modB, or modC gene. The addition of molybdate to the growth medium of a mod mutant lowered phi (modA'-'lacZ+) expression. Repression of the mod operon was sensitive to molybdate but was insensitive to mutations in the MGD synthetic pathway. These physiological and genetic experiments show that molybdate can be transported by one of the following three anion transport system in E. coli: the native system, the sulfate transport system (cysTWA gene products), and an undefined transporter. Upon entering the cytoplasm, molybdate branches out to mod regulation, fdhF and hyc activation, and metabolic conversion, leading to MGD synthesis and active molybdoenzyme synthesis.