Zinc coordination, function, and structure of zinc enzymes and other proteins

Addition of benzoate to the medium reservoir of glucose-limited chemostat cultures of Saccharomyces cerevisiae CBS 8066 growing at a dilution rate (D) of 0.10 h-1 resulted in a decrease in the biomass yield, and an increase in the specific oxygen uptake rate (qO2) from 2.5 to as high as 19.5 mmol g-1 h-1. Above a critical concentration, the presence of benzoate led to alcoholic fermentation and a reduction in qO2 to 13 mmol g-1 h-1. The stimulatory effect of benzoate on respiration was dependent on the dilution rate: at high dilution rates respiration was not enhanced by benzoate. Cells could only gradually adapt to growth in the presence of benzoate: a pulse of benzoate given directly to the culture resulted in wash-out. As the presence of benzoate in cultures growing at low dilution rates resulted in large changes in the catabolic glucose flux, it was of interest to study the effect of benzoate on the residual glucose concentration in the fermenter as well as on the level of some selected enzymes. At D = 0.10 h-1, the residual glucose concentration increased proportionally with increasing benzoate concentration. This suggests that modulation of the glucose flux mainly occurs via a change in the extracellular glucose concentration rather than by synthesis of an additional amount of carriers. Also various intracellular enzyme levels were not positively correlated with the rate of respiration. A notable exception was citrate synthase: its level increased with increasing respiration rate. Growth of S. cerevisiae in ethanol-limited cultures in the presence of benzoate also led to very high qO2 levels of 19-21 mmol g-1 h-1. During growth on glucose as well as on ethanol, the presence of benzoate coincided with an increase in the mitochondrial volume up to one quarter of the total cellular volume. Also with the Crabtree-negative yeasts Candida utilis, Kluyveromyces marxianus and Hansenula polymorpha, growth in the presence of benzoate resulted in an increase in qO2 and, at high concentrations of benzoate, in aerobic fermentation. In contrast to S. cerevisiae, the highest qO2 of these yeasts when growing at D = 0.10 h-1 in the presence of benzoate was equal to, or lower than the qO2 attainable at mu(max) without benzoate. Enzyme activities that were repressed by glucose in S. cerevisiae also declined in K. marxianus when the glucose flux was increased by the presence of benzoate.(ABSTRACT TRUNCATED AT 400 WORDS)

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Effect of benzoic acid on metabolic fluxes in yeasts: A continuous‐culture study on the regulation of respiration and alcoholic fermentation

Phosphate moieties bind frequently at N-termini of helices in proteins. It is shown that this corresponds with an optimal interaction of the helix dipole and the charged phosphate. This favourable arrangement may have been discovered several times during evolution. In some enzymes, the helix dipole might be used in catalysis.

Alpha-helix dipole and properties of proteins

The cell wall is essential to nearly every aspect of the biology and pathogenicity of Candida albicans. Although it was intially considered an almost inert cellular structure that protected the protoplast against osmotic offense, more recent studies have demonstrated that it is a dynamic organelle. The major components of the cell wall are glucan and chitin, which are associated with structural rigidity, and mannoproteins. The protein component, including both mannoprotein and nonmannoproteins, comprises some 40 or more moieties. Wall proteins may differ in their expression, secretion, or topological location within the wall structure. Proteins may be modified by glycosylation (primarily addition of mannose residues), phosphorylation, and ubiquitination. Among the secreted enzymes are those that are postulated to have substrates within the cell wall and those that find substrates in the extracellular environment. Cell wall proteins have been implicated in adhesion to host tissues and ligands. Fibrinogen, complement fragments, and several extracellular matrix components are among the host proteins bound by cell wall proteins. Proteins related to the hsp70 and hsp90 families of conserved stress proteins and some glycolytic enzyme proteins are also found in the cell wall, apparently as bona fide components. In addition, the expression of some proteins is associated with the morphological growth form of the fungus and may play a role in morphogenesis. Finally, surface mannoproteins are strong immunogens that trigger and modulate the host immune response during candidiasis.

Cell Wall and Secreted Proteins of Candida albicans: Identification, Function, and Expression

Short-chain dehydrogenases/reductases (SDRs) constitute a large family of NAD(P)(H)-dependent oxidoreductases, sharing sequence motifs and displaying similar mechanisms. SDR enzymes have critical roles in lipid, amino acid, carbohydrate, cofactor, hormone and xenobiotic metabolism as well as in redox sensor mechanisms. Sequence identities are low, and the most conserved feature is an alpha/beta folding pattern with a central beta sheet flanked by 2 - 3 alpha-helices from each side, thus a classical Rossmannfold motif for nucleotide binding. The conservation of this element and an active site, often with an Asn-Ser-Tyr-Lys tetrad, provides a platform for enzymatic activities encompassing several EC classes, including oxidoreductases, epimerases and lyases. The common mechanism is an underlying hydride and proton transfer involving the nicotinamide and typically an active site tyrosine residue, whereas substrate specificity is determined by a variable C-terminal segment. Relationships exist with bacterial haloalcohol dehalogenases, which lack cofactor binding but have the active site architecture, emphasizing the versatility of the basic fold in also generating hydride transfer-independent lyases. The conserved fold and nucleotide binding emphasize the role of SDRs as scaffolds for an NAD(P)(H) redox sensor system, of importance to control metabolic routes, transcription and signalling.

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Medium- and short-chain dehydrogenase/reductase gene and protein families: The SDR superfamily: functional and structural diversity within a family of metabolic and regulatory enzymes

Publisher Summary This chapter describes the advances with an emphasis on the structures of the alcohol dehydrogenases and the relationship between structure and function Yeast and mammalian alcohol dehydrogenase differ in substrate specificity and rate of catalytic activity The classic yeast enzyme is more specific for acetaldehyde and ethanol, which is consistent with its recognized physiological Significance to participate in alcohol fermentation at the end of the glycolytic pathway Enzyme forms with other functions and properties also occur in yeast The mammalian enzymes have broad substrate specificity and, even with primary alcohols, the maximum activity is not observed with ethanol Alcohols including ethanol, produced in the intestinal tracts mainly by bacterial actions, are found in the portal vein One physiological function of liver alcohol dehydrogenase may be to metabolize these products Structural studies have established that mammalian alcohol dehydrogenases have a distant evolutionary link to both the yeast and bacterial enzymes Ingested alcohol is metabolized to acetaldehyde mainly by the action of liver alcohol dehydrogenase

Bo Furugren

Papers

3 Alcohol Dehydrogenases