Carbon Monoxide Dehydrogenase (CODH), also known as Acetyl-CoA synthase (ACS), is one of seven known Ni containing enzymes. CODH/ACS is a bifunctional enzyme which oxidizes CO to CO2 reversibly and synthesizes acetyl-CoA. Recently, X-ray crystal structures of homodimeric CODH from Rhodospirillum rubrum (CODHRr) and CODH from Carboxydothermus hydrogenoformans (CODHCh) have been published. These two enzymes catalyze only the reversible oxidation of CO to CO2 and have a protein sequence homologous to that of the β subunit of heterotetrameric α2β2 enzyme from Moorella thermoacetica (CODHMt), formerly Clostridium thermoaceticum. Neither CODHRr nor CODHCh contain an α-subunit as is found in CODHMt. The precise structure of the active site for acetyl-CoA synthase, called the A-cluster, is not known. Therefore, crystallization of the α subunit is required to solve the remaining structural features of CODH/ACS. Obtaining crystals and determining the X-ray crystal structure is a high-risk endeavor, and a second project was pursued involving the preparation, expression and analysis of various site-directed mutants of CODHMt. Mutational analysis of particular histidine residues and various other conserved residues of CODH from Moorella thermoacetica is discussed. Visual inspection of the crystal structure of CODHRr and CODHCh, along with sequence alignments, indicates that there may be separate pathways for proton and electron transfer during catalysis. Mutants of a proposed proton transfer pathway were characterized. Four semi-conserved histidine residues were individually mutated to alanine. Two (His116Mt and His122Mt) were essential to catalysis, while the other two (His113Mt and His119Mt) attenuated catalysis but were not essential. Significant activity was "rescued" by a double mutant where His116 was replaced by Ala and His was also introduced at position 115. Activity was also rescued in double mutants where His122 was replaced by Ala and His was simultaneously introduced at either position 121 or 123. Activity was also "rescued" by replacing His with Cys at position 116. Mutation of conserved Lys587 near the C-cluster attenuated activity but did not eliminate it. Activity was virtually abolished in a double mutant where Lys587 and His113 were both changed to Ala. Mutations of conserved Asn284 also attenuated activity. These effects suggest the presence of a network of amino acid residues responsible for proton transfer rather than a single linear pathway.

Legionella pneumophila, the causative agent of Legionnaires’ disease (LD), is an intracellular pathogen of freshwater protozoa that can also persist in the environment as a free-living bacterium. L. pneumophila has many morphological forms that fit within a developmental cycle. In water, L. pneumophila enters into a viable but non-culturable (VBNC) state that is largely uncharacterized. VBNC cells were produced from two developmental L. pneumophila forms, stationary phase forms (SPFs) and mature infectious forms (MIFs) by suspension in double deionized (dd) or tap-water at 45°C. Electron microscopy results showed that VBNC cells have a unique morphology and that in tap water they lose their poly 3-hydroxybutyrate inclusion bodies. Both SPFs and MIFs lost culturability faster in dd- than in tap water, and addition of salts to dd-water prolonged L. pneumophila culturability and enhanced viability. However, MIFs retained higher viability in dd- and tap water (85% and 51%, respectively) than SPFs (5% and 20%, respectively) as determined by the BacLight vital stain. Only ~1 VBNC cell out of 105 of those produced from SPFs in tap water regained culturability via infection of Acanthamoeba. All VBNC cells, except for those produced from SPFs in dd-water, resisted both digestion inside Tetrahymena spp. and detergent-mediated lysis. SDS-PAGE analysis and shotgun proteomics revealed a number of VBNC cell specific proteins; one of these was 3-hydroxybutyrate dehydrogenase (BdhA), which is involved in the metabolism of poly 3-hydroxybutyrate inclusion bodies. A bdhA mutant showed an early loss of culturability and a dramatic decrease in viability as compared to the parent strain, and complementing the mutant with a functional bdhA gene restored the parent's strain phenotypes. In conclusion, VBNC L. pneumophila has a distinct morphology and physiology that varies according to the developmental stage and the environmental conditions used to produce such VBNC cells. VBNC cells have a different protein profile and morphology than the culturable cells, suggesting that this state constitutes a distinct differentiated form within the developmental cycle of L. pneumophila. BdhA seems to influence L. pneumophila survival and hence VBNC cell formation. Collectively, the results from this study provide a better understanding of L. pneumophila VBNC form and the factors influencing its formation.

The aldolase-dehydrogenase complex catalyzes the last two steps in the microbial meta-cleavage pathway of various aromatic compounds including polychlorinated biphenyls (bph pathway) and cholesterol (hsa pathway). The aldolase, BphI, cleaves 4-hydroxy-2-oxoacids to produce pyruvate and an aldehyde. Linear aldehydes of up to six carbons long and branched isobutyraldehyde were directly channeled to the aldehyde dehydrogenase BphJ, via a molecular tunnel, with greater than 80% efficiency. The molecular tunnel is narrow in positions lined by Gly-322 and Gly-323 in the aldolase. BphI variants G322F, G322L and G323F were found to block aldehyde channeling. The replacement of Asn-170 in BphJ with alanine and aspartate did not substantially alter aldehyde channeling efficiencies, thus disproving a previous hypothesis that hydrogen bonding between the Asn-170 and the nicotinamide cofactor induces the opening of the exit of the tunnel. The H20A and Y290F BphI variants displayed significantly reduced aldehyde channeling efficiencies indicating that these residues control the entry and exit of substrates and products from the aldolase reaction. The BphI reaction was activated by NADH binding to BphJ in the wild-type enzyme and channel blocked variants. Activation of BphI by BphJ N170A, N170D and I171A was decreased by ≥ 3-fold in the presence of NADH and ≥ 4.5-fold when BphJ was undergoing turnover. These results demonstrate that the dehydrogenase coordinates catalytic activity of BphI through allostery rather than through faster aldehyde release from substrate channeling. HsaF, an ortholog of BphI from Mycobacterium tuberculosis could be expressed as a soluble dimer, however HsaF was inactive in the absence of HsaG, a BphJ ortholog. Acetaldehyde and propionaldehyde were channeled directly to HsaG with similar efficiencies as in the BphI-BphJ system. The HsaF-HsaG complex was crystallized and its structure solved to a resolution of 1.93 Å. Substitution of Ser-41 in HsaG with isoleucine or aspartate resulted in about 35-fold increase in Km for CoA but only 4-fold increase in Km for dephospho-CoA, confirming its importance in interacting with the 3’- ribose phosphate of CoA. A second gene annotated as 4-hydroxy-2-oxopentanoic acid aldolase (Rv3469c) from M. tuberculosis was expressed, purified and found to possess oxaloacetate decarboxylase and not aldolase activity.

A Sinorhizobium meliloti (S. meliloti ) mutant, Rm30O49, deficient in malate dehydrogenase (MDH) activity was isolated via random Tn5tac1 mutagenesis. DNA sequence analyses revealed 60 the inaction is within the mdh gene. Rm30049 lacks MDH activity under all growth conditions, but shows increased or decreased activities of the TCA cycle enzymes 2-oxoglutarate dehydrogenase and succinate dehydrogenase in the presence or absence, respectively, of IPTG (isopropyl beta-D-thiogalactoside). The symbiotic phenotype of the mutant is an inability to fix nitrogen. Alfalfa seedlings inoculated with Rm30049 produced small white root nodules, but were chlorotic and failed to reach a wild-type shoot dry weight. Cosmid clone pDS15 was isolated by heterologous complementation of a Rhizobium leguminosarum sucD mutant by the S. meliloti pLAFR1 clone bank. This cosmid also restored MDH activity to Rm30049, and complemented the mutant growth and symbiotic phenotypes. Three Tn5 insertions isolated in pDS15 within sucA failed to complement Rm30049. DNA sequence analyses indicate that the mdh gene is part of the TCA cycle operon with sucCD, and that downstream and upstream of this, are operons encoding sucAB and sdhCDAB, respectively.

Hyperthermophiles are microorganisms that can grow at temperatures close to the boiling point of water or above. They are potential resources of thermostable enzymes including alcohol dehydrogenases (ADHs). Both Thermococcus guaymasensis and Thermotoga hypogea produce ethanol as an end product using glucose as substrate. However, the metabolic pathway and enzymes involved in alcohol production by these hyperthermophiles were not clear. ADH is a key enzyme responsible for alcohol metabolism, and the enzyme has been purified and characterized. T. hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90??C. The NADP+-dependent ADH from T. hypogea was purified to homogeneity and a homodimeric protein with a subunit size of 40 ?? 1 kDa. A part of its encoding gene was cloned and sequenced, from which a major part of the amino acid sequence of the enzyme was deduced and found to have high similarities to iron-containing ADHs from other Thermotoga species and harbored typical iron and NADP+-binding motifs. The conserved domain search showed that T. hypogea ADH was a member of the family of uncharacterized iron-containing ADHs. The iron content of the enzyme was determined to be 1.02 ?? 0.06 g-atoms per subunit. It is the first characterized iron-containing ADH from hyperthermophilic bacteria. Similar to known iron-containing ADHs, T. hypogea ADH was oxygen sensitive; however, the loss of enzyme activity upon exposure to oxygen could be recovered by incubation with dithiothreitol and Fe2+. The enzyme was thermostable with a half-life of about 10 h at 70??C, and its catalytic activity increased along with the rise of temperatures up to 95??C. Optimal pH values for the production and oxidation of alcohol were determined to be 8.0 and 11.0, respectively. The enzyme had a broad specificity in utilizing primary alcohols and aldehydes as substrates. Apparent Km values for ethanol and 1-butanol were much higher than that for acetaldehyde and butyraldehyde and thus the enzyme was likely to catalyze the reduction of aldehydes to alcohols in vivo. T. guaymasensis is a hyperthermophilic anaerobic archaeon capable of catalyzing the starch degradation and producing ethanol and acetoin as end-products. The purified T. guaymasensis ADH was an NADP+-dependent homotetramer with a subunit of 40 ?? 1 kDa. The enzyme was a primary-secondary ADH, but it exhibited substrate preference on secondary alcohols and corresponding ketones. In particular, it catalyzed the reduction of diacetyl to 2, 3-butanediol via acetoin in which the reduction from diacetyl to acetoin was irreversible. For the oxidation of 2, 3-butanediol, the enzyme exhibited higher activities on (2R, 3R)-(-)-2, 3-butanediol and meso-2, 3-butanediol than (2S, 3S)-(+)-2, 3-butanediol while the stereoselective reduction of racemic (R/S)-acetoin produced (2R, 3R)-(-)-2, 3-butanediol and meso-butanediol but not (2S, 3S)-(+)-2, 3-butanediol. The optimal pHs for the oxidation and formation of alcohols were determined to be 10.5 and 7.5, respectively. The enzyme activity increased along with the rise of temperatures up to 95??C, and it was highly stable with a half-life of 24 hours at 95??C. The enzyme was resistant to 30% (v/v) methanol (retaining 40% of its full activity). NADPH for the ketone reduction was efficiently regenerated using isopropanol as a substrate. The apparent Km value for NADPH was 40 times lower than that of NADP+, and the specificity constant with NADPH were 5 times higher than that of NADP+. Therefore, the physiological role of the enzyme was likely to be responsible for the reactions involving the NADPH oxidation???coupled formation of ethanol and/or acetoin. The fully active T. guaymasensis ADH contained 0.9 ?? 0.03 g atom zinc per subunit determined by inductively coupled plasma mass spectrometry (ICP-MS) and was the first characterized zinc-containing ADH from Thermococcus species. The gene encoding this enzyme was cloned and sequenced, and the deduced amino acid sequence contained 364 amino acids showing high similarities (85%) to those ADHs from Thermoanaerobacter species which have only the catalytic zinc atom. The motif analyses also indicated the enzyme lacked of the structural zinc-binding motif; thus, zinc might play a catalytic role in the enzyme. Further analyses showed the presence of the conserved domains of L-threonine dehydrogenases; however, the enzyme could not oxidize L-threonine or L-serine. Distinct from most of zinc-containing ADHs, the enzyme activity was almost fully inhibited by 100 ??M Zn2+ in the assay mixture. Moreover, it was sensitive to oxygen. An NADP+-dependent ADH was purified from the hyperthermophilic anaerobic archaeon Thermococcus strain ES1, an ethanol producer. The recombinant enzyme over-expressed in Escherichia coli was purified using a two-step procedure including heat treatment, and characterized in comparison with the native enzyme. The purified recombinant enzyme exhibited a specific activity of 52.8 U mg-1, close to that of the native enzyme (57 U mg-1). Both native and recombinant enzymes were homotetramers with a subunit size of 45 ?? 1 kDa. Their optimal pHs for the ethanol oxidation and acetaldehyde reduction were determined to be 10.5 and 7.0, respectively. Both enzymes were able to oxidize a series of primary alcohols and diols. Metal contents of the fully active recombinant enzyme were determined by ICP-MS to be 1.0 ?? 0.04 g atom iron per subunit, and both iron-containing enzymes were oxygen sensitive. Their kinetic parameters showed lower Km-values of acetaldehyde and NADPH than those of ethanol and NADP+, suggesting the native enzyme could be involved in ethanol formation in vivo. The recombinant and native enzymes had almost identical characteristics and thus its encoding gene was successfully over-expressed in E. coli. The deduced amino acid sequence of the ADH from Thermococcus strain ES1 was a 406 amino acid polypeptide. Its amino acid sequence showed high identities (80%) to iron-containing ADHs from the related archaea Thermococcus zilligii and Thermococcus hydrothermalis. The conserved domain search revealed it belonged to the family of iron-containing ADHs. Moreover, the sequence of the enzyme had catalytic metal and dinucleotide-binding motifs typical for iron-containing ADHs. In conclusion, the results indicate that iron- and zinc-containing ADHs from hyperthermophiles have significant differences in terms of biophysical, biochemical and molecular properties. The hyperthermophilic bacterial and archaeal iron-containing ADHs are divergent while the zinc-containing ADH from T. guaymasensis has significant similarity to thermophilic bacterial ones.

No description available.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2002. / Härtill 6 uppsatser.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2006. / Vita. Includes bibliographical references.

Thesis (M.Sc. (Biochemistry)) -- Mahidol University, 1976.

Thesis (M. Sc.)--Carleton University, 2008. / Includes bibliographical references (p. 116-125). Also available in electronic format on the Internet.