Stephanie Bringer-Meyer

Pyruvate decarboxylase from Zymomonas mobilis. Isolation and partial characterization

Pyruvate decarboxylase (EC 4.1.1.1) from the ethanol producing bacterium Zymomonas mobilis was purified to homogeneity. This enzyme is an acidic protein with an isoelectric point of 4.87 and has an apparent molecular weight of 200,000±10,000. The enzyme showed a single band in sodium dodecylsulfate gel electrophoresis with a molecular weight of 56,500±4,000 which indicated that the enzyme consists of four probably identical subunits. The dissociation of the cofactors Mg2+ and thiamine pyrophosphate at pH 8.9 resulted in a total loss of enzyme activity which could be restored to 99.5% at pH 6.0 in the presence of both cofactors. For the apoenzyme the apparent Km values for Mg2+ and thiamine pyrophosphate were determined to be 24 μM and 1.28 μM. The apparent Km value for the substrate pyruvate was 0.4 mM. Antiserum prepared against this purified pyruvate decarboxylase failed to crossreact with cell extracts of the reportedly pyruvate decarboxylase positive bacteria Sarcina ventriculi, Erwinia amylovora, or Gluconobacter oxydans, or with cell extracts of Saccharomyces cerevisiae.

Enantioselective reduction of carbonyl compounds by whole-cell biotransformation, combining a formate dehydrogenase and a (R)-specific alcohol dehydrogenase

A whole-cell biotransformation system for the reduction of prochiral carbonyl compounds, such as methyl acetoacetate, to chiral hydroxy acid derivatives [methyl (R)-3-hydroxy butanoate] was developed in Escherichia coli by construction of a recombinant oxidation/reduction cycle. Alcohol dehydrogenase from Lactobacillus brevis catalyzes a highly regioselective and enantioselective reduction of several ketones or keto acid derivatives to chiral alcohols or hydroxy acid esters. The adh gene encoding for the alcohol dehydrogenase of L. brevis was expressed in E. coli. As expected, whole cells of the recombinant strain produced only low quantities of methyl (R)-3-hydroxy butanoate from the substrate methyl acetoacetate. Therefore, the fdh gene from Mycobacterium vaccae N10, encoding NAD+-dependent formate dehydrogenase, was functionally coexpressed. The resulting two-fold recombinant strain exhibited an in vitro catalytic alcohol dehydrogenase activity of 6.5 units mg−1 protein in reducing methyl acetoacetate to methyl (R)-3-hydroxy butanoate with NADPH as the cofactor and 0.7 units mg−1 protein with NADH. The in vitro formate dehydrogenase activity was 1.3 units mg−1 protein. Whole resting cells of this strain catalyzed the formation of 40 mM methyl (R)-3-hydroxy butanoate from methyl acetoacetate. The product yield was 100 mol% at a productivity of 200 μmol g−1 (cell dry weight) min−1. In the presence of formate, the intracellular [NADH]/[NAD+] ratio of the cells increased seven-fold. Thus, the functional overexpression of alcohol dehydrogenase in the presence of formate dehydrogenase was sufficient to enable and sustain the desired reduction reaction via the relatively low specific activity of alcohol dehydrogenase with NADH, instead of NADPH, as a cofactor.

Zymomonas mobilis mutants blocked in fructose utilization

SummaryA mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.

Biochemistry and physiology of hopanoids in bacteria.

Publisher Summary Hopanoids are amphipathic components of bacterial cell membranes and maintain membrane stability by increasing rigidity in the lipid matrix. Thus, the function of hopanoids in bacteria is analogous to that of membrane sterols in eukaryotes. Biosynthesis of hopanoids does not require oxygen during squalene cyclization and other reactions. This chapter describes the structures, distribution, and physiological role of hopanoids as membrane stabilizers. Structural variations in hopanoids result from linkage between a hydrophobic triterpenic moiety and a polar side-chain. The simplest hopanoids, diplopterol and diploptene, are readily extractable from freeze-dried cells by organic solvents and can be characterized by gas-liquid chromatography (GLC) and by combined GLC-mass spectroscopy. Another methods for analysis of periodate-oxidized and borohydride-reduced hopanoids, where hopanoid alcohols are converted into benzoate or naphthoate derivatives, yielding chromogenic compounds that can be separated with a reversed-phase high-pressure liquid chromatography (HPLC) system using ultraviolet-radiation detection are presented. Hopanoid-containing fractions can usually be detected only by proton nuclear magnetic-resonance (NMR) spectroscopy exploiting the characteristic pattern of methyl singlets in the hopane skeleton. The occurrence of hopanoids in the specialized organisms exemplifies their general role as membrane reinforcers in eubacteria. The presence of hopanoids is essential for survival of the bacteria that contain them. The chapter accounts for the biosynthesis and principles of genetics for the hopanoids. Hopanoids are abundant natural products of eubacteria. The lipid family of hopanoids consists of numerous structural variants that lead to their detection and analysis.

GENE AND SUBUNIT ORGANIZATION OF BACTERIAL PYRUVATE DEHYDROGENASE COMPLEXES

Pyruvate dehydrogenase complexes of bacterial origin are compared with respect to subunit composition, organization of the corresponding genes, and the number and location of lipoyl domains. Special attention is given to two unusual examples of pyruvate dehydrogenase complexes, formed by Zymomonas mobilis and Thiobacillus ferrooxidans.

Effect of alcohols and temperature on the hopanoid content of Zymomonas mobilis

SummaryThe influence of different culture conditions on the hopanoid content of Zymomonas mobilis was investigated in batch cultures. With a gas-liquid chromatographic method it could be shown that the content of 1,2,3,4-tetrahydroxypentane-29-hopane (THBH) reached a maximum value in the stationary phase due to the high level of ethanol accumulated in the medium. The hopanoid content increased sharply with the addition of ethanol to the culture. Ethanol was shown to be the most effective of the alcohols tested in causing an increase of the hopanoid content. Furthermore, an alteration of the incubation temperature from 14° to 37°C also caused an increase of the amount of hopanoids.

Oxidative phosphorylation in Zymomonas mobilis

The obligately fermentative aerotolerant bacterium Zymomonas mobilis was shown to possess oxidative phosphorylation activity. Increased intracellular ATP levels were observed in aerated starved cell suspension in the presence of ethanol or acetaldehyde. Ethanolconsuming Z. mobilis generated a transmembrane pH gradient. ATP synthesis in starved Z. mobilis cells could be induced by external medium acidification of 3.5–4.0 pH units. Membrane vesicles of Z. mobilis coupled ATP synthesis to NADH oxidation. ATP synthesis was sensitive to the protonophoric uncoupler CCCP both in starved cells and in membrane vesicles. The H+-ATPase inhibitor DCCD was shown to inhibit the NADH-coupled ATP synthesis in membrane vesicles. The physiological role of oxidative phosphorylation in this obligately fermentative bacterium is discussed.

Osmotic adjustment of Zymomonas mobilis to concentrated glucose solutions

SummaryThe physiological basis of the exceptionally high sugar tolerance of Zymomonas mobilis was investigated. Determinations of the internal metabolite concentrations of Z. mobilis showed that an increase in the extracellular glucose concentration was accompanied by a parallel rise in the intracellular glucose concentration, bringing about an almost complete osmotic balance between internal and external space. Studies of glucose transport confirmed that Z. mobilis has a facilitated diffusion system which enables a rapid equilibration between internal and external glucose concentrations. Studies using the non-metabolisable sugars maltose (impermeable) and xylose (permeable) revealed that these sugars were able to alter the osmotic pressure on the cytoplasmic membrane resulting in volume changes.

Glucose oxidation and PQQ-dependent dehydrogenases in Gluconobacter oxydans.

Gluconobacter oxydans is famous for its rapid and incomplete oxidation of a wide range of sugars and sugar alcohols. The organism is known for its efficient oxidation of D-glucose to D-gluconate, which can be further oxidized to two different keto-D-gluconates, 2-keto-D-gluconate and 5-keto-D-gluconate, as well as 2,5-di-keto-D-gluconate. For this oxidation chain and for further oxidation reactions, G. oxydans possesses a high number of membrane-bound dehydrogenases. In this review, we focus on the dehydrogenases involved in D-glucose oxidation and the products formed during this process. As some of the involved dehydrogenases contain pyrroloquinoline quinone (PQQ) as a cofactor, also PQQ synthesis is reviewed. Finally, we will give an overview of further PQQ-dependent dehydrogenases and discuss their functions in G. oxydans ATCC 621H (DSM 2343).

Formation and degradation of gluconate by Zymomonas mobilis

SummaryZymomonas mobilis ATCC 29191 is able to degrade gluconate but cannot use it as a single carbon and energy source. Gluconate is phosphorylated by a gluconate kinase (EC 2.7.1.12) and the resulting 6-phosphogluconate is further catabolized to yield about 0.8 mol ethanol per mol of gluconate, considerable amounts of acetate and acetoin. This product spectrum agrees with the theoretical yield of only one reduction equivalent if gluconate is phosphorylated by a kinase and subsequently metabolized via the Entner-Doudoroff pathway.Furthermore, Z. mobilis contains a membrane-bound enzyme system which is able to oxidize glucose to gluconate. Cell-free extracts were active in an assay system with Wursters blue as electron acceptor, and various aldoses as well as maltose, mannitol and sorbitol could be oxidized. The affinity for sorbitol was very low (Km=330 mM) but reasonable for glucose (Km=2.8 mM). The pH optimum for the glucose-oxidizing reaction was 6.5, while that for sorbitol oxidation was 5.5.