Comparative study of the activity and kinetic properties of malate dehydrogenase and pyruvate decarboxylase from Candida albicans, Malassezia pachydermatis, and Saccharomyces cerevisiae

Candida albicans and Malassezia pachydermatis cause human and animal infections of the skin and internal organs. We compare the properties of two enzymes, pyruvate decarboxylase (PDC) and malate dehydrogenase (MDH), from these species and from Saccharomyces cerevisiae cultivated under aerobic and anaerobic conditions to find differences between the enzymes that adapt pathogens for virulence and help us in searching for new antifungal agents. Malassezia pachydermatis did not show any growth under anaerobic conditions, as opposed to C. albicans and S. cerevisiae. Under aerobic conditions, C. albicans showed the highest growth rate. Malassezia pachydermatis, contrary to the others, did not show any PDC activity, simultaneously showing the highest MDH activity under aerobic conditions and a Km value for oxaloacetate lower than S. cerevisiae. Candida albicans and S. cerevisiae showed a strong decrease in MDH activity under anaerobic conditions. Candida albicans shows four different isoforms of MDH, while M. pachydermatis and S. cerevisiae are characterized by two and three isoforms. Candida albicans shows about a twofold lower activity of PDC but, simultaneously, almost a threefold lower Km value for pyruvate in comparison with S. cerevisiae. The PDC apoform share under aerobic conditions in C. albicans was 47%, while in S. cerevisiae was only 26%; under anaerobic conditions, the PDC apoform decreased to 12% and 8%, respectively. The properties of enzymes from C. albicans show its high metabolic flexibility (contrary to M. pachydermatis) and cause easy switching between fermentative and oxidative metabolism. This feature allows C. albicans to cause both surface and deep infections. We take into consideration the use of thiamin antimetabolites as antifungal factors that can affect both oxidative and fermentative metabolism.     

Conversion of coconut oil to methyl ketones by two Aspergillus species

Isolates of Aspergillus ruber and A. repens have been grown on coconut oil as the sole carbon source in shake culture. Methyl ketones (C₅-C₁₃) were isolated by solvent extraction and analysed by combined gas chromatography and mass spectrometry. 2-Undecanone was the main volatile product reflecting the high concentration of dodecanoic acid in the original coconut oil. The reactivity of the individual short chain fatty acids as substrates for production of methyl ketones would appear to decrease with increasing molecular weight of the acid after taking into account the greater volatility of the lower molecular weight homologues. 2-Hexanone and 2-octanone were produced by all isolates in low concentration (< 1%). Nonanoic acid and 2-heptanone were converted into 2-octanone. Low concentrations of secondary alcohols were formed under aerobic conditions. It is suggested that the production of methyl ketones by partial β-oxidation is too closely related to mainstream metabolism to be of use in the biochemical taxonomy of the genus.     

Pyruvate decarboxylases from the petite-negative yeast<Emphasis Type="Italic"> Saccharomyces kluyveri</Emphasis>

Saccharomyces kluyveri is a petite-negative yeast, which is less prone to form ethanol under aerobic conditions than is S. cerevisiae. The first reaction on the route from pyruvate to ethanol is catalysed by pyruvate decarboxylase, and the differences observed between S. kluyveri and S. cerevisiae with respect to ethanol formation under aerobic conditions could be caused by differences in the regulation of this enzyme activity. We have identified and cloned three genes encoding functional pyruvate decarboxylase enzymes ( PDC genes) from the type strain of S. kluyveri (Sk-PDC11, Sk-PDC12 and Sk-PDC13). The regulation of pyruvate decarboxylase in S. kluyveri was studied by measuring the total level of Sk-PDC mRNA and the overall enzyme activity under various growth conditions. It was found that the level of Sk-PDC mRNA was enhanced by glucose and oxygen limitation, and that the level of enzyme activity was controlled by variations in the amount of mRNA. The mRNA level and the pyruvate decarboxylase activity responded to anaerobiosis and growth on different carbon sources in essentially the same fashion as in S. cerevisiae. This indicates that the difference in ethanol formation between these two yeasts is not due to differences in the regulation of pyruvate decarboxylase(s), but rather to differences in the regulation of the TCA cycle and the respiratory machinery. However, the PDC genes of Saccharomyces/Kluyveromyces yeasts differ in their genetic organization and phylogenetic origin. While S. cerevisiae and S. kluyveri each have three PDC genes, these have apparently arisen by independent duplications and specializations in each of the two yeast lineages.Communicated by C. P. Hollenberg     

Highly efficient selective oxidation of alcohols to carbonyl compounds catalyzed by ruthenium (III) meso-tetraphenylporphyrin chloride in the presence of molecular oxygen

Efficient selective oxidation of alcohols to carbonyl compounds by molecular oxygen with isobutyraldehyde as oxygen acceptor in the presence of metalloporphyrins has been reported. Ruthenium (III) meso-tetraphenylporphyrin chloride (Ru(TPP)Cl) showed excellent activity and selectivity for oxidation of various alcohols under mild conditions. Moreover, different factors influencing alcohols oxidation, for example, catalyst, solvent, temperature, and oxidant, have been investigated. In large-scale oxidation of benzyl alcohol, the isolated yield of benzaldehyde of 89% was observed.     

The pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses

Ethanolic fermentation is classically associated with flooding tolerance when plant cells switch from respiration to anaerobic fermentation. However, recent studies have suggested that fermentation also has important functions in the presence of oxygen, mainly in germinating pollen and during abiotic stress. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we characterize the PDC gene family in Arabidopsis. PDC is encoded by four closely related genes. By using real-time quantitative polymerase chain reaction, we determined the expression levels of each individual gene in different tissues, under normal growth conditions, and when the plants were subjected to anoxia or other environmental stress conditions. We show that PDC1 is the only gene induced under oxygen limitation among the PDC1 gene family and that a pdc1 null mutant is comprised in anoxia tolerance but not other environmental stresses. We also characterize the expression of the aldehyde dehydrogenase (ALDH) gene family. None of the three genes is induced by anoxia but ALDH2B7 reacts strongly to ABA application and dehydration, suggesting that ALDH may play a role in aerobic detoxification of acetaldehyde. We discuss the possible role of ethanolic fermentation as a robust back-up energy production pathway under adverse conditions when mitochondrial function is disturbed.     

Denaturation of Aplysia myoglobin. Equilibrium study

The effect of temperature and solvent composition on the reversible denaturation of Aplysia myoglobin is reported here. The denaturation is accompanied by changes in the haem absorption spectrum, in the intrinsic fluorescence of the protein and in its optical activity in the Soret and ultraviolet regions. Under all conditions examined (i.e. different temperatures, different concentrations of various alcohols and different concentrations of proteins) the system appears to be in a state of true equilibrium and good agreement is found between thermally-induced and solvent-induced transitions.     

Formation of coniferyl alcohol from ferulic acid by the white rot fungus Trametes

The white rot fungus, Trametes sp., was cultivated in a medium containing ferulic acid, glucose and ethanol under aerobic conditions in submerged culture. The ferulic acid was transformed into coniferyl alcohol, coniferylaldehyde, dihydroconiferyl alcohol, vanillic acid, vanillyl alcohol, 2-methoxyhydroquinone and 2-methoxyquinone during 48–120 hr of cultivation. The amount of coniferyl alcohol in the culture reached a maximum after 90 hr with ca 40% of the initial amount of ferulic acid. Cinnamic acid, p-methoxycinnamic acid, 3,4-dimethoxycinnamic acid, p -coumaric acid and sinapic acid were also transformed into the corresponding alcohols, benzoic acids and benzyl alcohols in the fungus culture.     

Highly efficient controllable oxidation of alcohols to aldehydes and acids with sodium periodate catalyzed by water-soluble metalloporphyrins as biomimetic catalyst

Highly efficient controllable oxidation of alcohols to aldehydes or acids by sodium periodate in the presence of water-soluble manganese porphyrins (meso-tetrakis(N-ethylpyridinium-4-yl)manganese porphyrin, MnTEPyP) with different reaction media has been reported. The manganese porphyrin showed excellent activity for the controllable oxidation of various alcohols under mild conditions. Moreover, different factors influencing alcohol oxidation, for example, oxidant, catalyst amount, temperature, and solvent, have been investigated. A plausible mechanism for the controllable oxidation of alcohol has been proposed.     

Facile Method for Monitoring Inhibition of Anaerobic Spore Outgrowth

A device is presented for the laboratory monitoring of spore outgrowth under controlled temperature and anaerobic conditions. Alterations in pH, redox potential, headspace composition, and optical density are followed as the activated spores grow out into vegetative cells. An interlock system allows the addition of test solutions or the removal of medium under anaerobic conditions. The device may also be used for rapid (<4 h) chemical inhibition studies or adapted for temperature injury studies of aerobic or anaerobic cells. Data on outgrowth of Clostridium sporogenes and inhibition by nitrite solutions are presented.     

The molecular mechanism of the temperature enhancement of proton magnetic relaxation rates in methaemoprotein solutions

The mechanism of water exchange between the haem-pocket and bulk solvent in aqueous methaemopiotein solutions was firmly substantiated by using the aliphatic protons of certain lower alcohols in an otherwise deuterated solution for measuring the incremental relaxation rates resulting from their magnetic interaction with the haem-iron. The fast-exchange condition was established for solutions of horse fluorometmyoglobin, human A fluoromethaemoglobin and Chironomus thummi aquomethaemoglobin. The distances between the exchangeable protons and the haem-iron obtained from these PMR measurements concur with the presence of the fluoride ion, while for Chironomus aquomethaemoglobin this distance is also much larger than that resulting from the location of the 6th site Water molecule. The latter finding is the first clear-cut evidence that the exchanging protons belong to the next neighbour water molecule, a previously advanced hypothesis. The exchanging water molecule may thus serve as a natural probe for comparing the haem-pocket conformational state(s) under different conditions or in various haemoproteins.     

Genetic and biochemical analysis of anaerobically-induced enzymes during seed germination of Echinochloa crus-galli varieties tolerant and intolerant of anoxia

To compare the regulation of anaerobic metabolism during germination in anoxia-tolerant and intolerant plants, enzymes associated with anaerobic metabolism such as sucrose synthase, aldolase, enolase, pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH) were assayed in two varieties of Echinochloa crus-galli, formosensis (tolerant) and praticola (intolerant). The initial and intervening enzymes of the pathway (sucrose synthase and aldolase) and enzymes in the last part of the pathway (PDC, ADH and ALDH) revealed similar changing patterns in activities during germination. This implies that each group of enzymes may be controlled by an identical regulatory mechanism. During anoxia, activities of all enzymes increased 1.5-30-fold in both varieties compared to their activities under aerobic conditions. Activities of sucrose synthase, enolase and ADH exhibited the same induction patterns under anoxia in formosensis and praticola. However, the activities of aldolase, ALDH and PDC were more strongly induced in formosensis under anoxia (1.2-2-fold) than in praticola. These enzymes were also assayed in F(3) families which varied in their anaerobic germinability. For PDC, activities under anoxia in anoxia-tolerant families were similar to those of an anoxia-intolerant family during the whole period although the family did not exhibit anaerobic germinability. This suggests that there is no correlation between PDC activity and anaerobic germinability. For ALDH, activities were more strongly induced under anoxia in anoxia-tolerant families than in anoxia-intolerant families, a trend also exhibited by the parents. This indicates that ALDH may play a role in detoxifying acetaldehyde formed through alcoholic fermentation during anaerobic germination.     

The impact of O(2) on the Fe-S cluster biogenesis requirements of Escherichia coli FNR

In this study, the functions of two established Fe-S cluster biogenesis pathways, Isc (iron-sulfur cluster) and Suf (sulfur mobilization), under aerobic and anaerobic growth conditions were compared by measuring the activity of the Escherichia coli global anaerobic regulator FNR. A [4Fe-4S] cluster is required for FNR activity under anaerobic conditions. An assay of the expression of FNR-dependent promoters in strains containing various deletions of the iscSUAhscBAfdx operon revealed that, under anaerobic conditions, FNR activity was reduced by 60% in the absence of the Isc pathway. In contrast, a mutant lacking the entire Suf pathway had normal FNR activity, although overexpression of the suf operon fully rescued the anaerobic defect in FNR activity in strains lacking the Isc pathway. Expression of the sufA promoter and levels of SufD protein were upregulated by twofold to threefold in Isc ⁻ strains under anaerobic conditions, suggesting that increased expression of the Suf pathway may be partially responsible for the FNR activity remaining in strains lacking the Isc pathway. In contrast, use of the O₂-stable [4Fe-4S] cluster FNR variant FNR-L28H showed that overexpression of the suf operon did not restore FNR activity to strains lacking the Isc pathway under aerobic conditions. In addition, FNR-L28H activity was more impaired under aerobic conditions than under anaerobic conditions. The greater requirement for the Isc pathway under aerobic conditions was not due to a change in the rate of Fe-S cluster acquisition by FNR-L28H under aerobic and anaerobic conditions, as shown by ⁵⁵Fe-labeling experiments. Using [³⁵S]methionine pulse-chase assays, we observed that the Isc pathway, but not the Suf pathway, is the major pathway required for conversion of O₂-inactivated apo-FNR into [4Fe-4S]FNR upon the onset of anaerobic growth conditions. Taken together, these findings indicate a major role for the Isc pathway in FNR Fe-S cluster biogenesis under both aerobic and anaerobic conditions.     

GroESL overexpression imparts Escherichia coli tolerance to i-, n-, and 2-butanol, 1,2,4-butanetriol and ethanol with complex and unpredictable patterns

Strain tolerance to toxic metabolites remains a limiting issue in the production of chemicals and biofuels using biological processes. Here we examined the impact of overexpressing the autologous GroESL chaperone system with its natural promoter on the tolerance of Escherichia coli to several toxic alcohols. Strain tolerance was examined using both a growth assay as well as viable cell counts employing a CFU (colony-forming unit) assay. GroESL over expression enhanced cell growth to all alcohols tested, including a 12-fold increase in total growth in 48-h cultures under 4% (v/v) ethanol, a 2.8-fold increase under 0.75% (v/v) n-butanol, a 3-fold increase under 1.25% (v/v) 2-butanol, and a 4-fold increase under 20% (v/v) 1,2,4-butanetriol. GroESL overexpression resulted in a 9-fold increase in CFU numbers compared to a plasmid control strain after 24 h of culture under 6% (v/v) ethanol, and a 3.5-fold and 9-fold increase for culture under 1% (v/v) n-butanol and i-butanol, respectively. The toxicity of the alcohols was examined against their octanol–water partition coefficient, a measure commonly used to predict solvent toxicity. For both the control and the GroESL overexpressing strains, the calculated membrane concentration of each alcohol based on the octanol–water partition coefficient could be correlated, but with different patterns, to the impact of the various alcohols on cell growth, but not on cell viability (CFUs). Our data suggest a complex pattern of growth inhibition and differential protection by GroESL overexpression depending on the specific alcohol molecule. Overall, however, GroESL overexpression appears to provide molecule-agnostic tolerance to toxic chemicals.     

Deletion of pyruvate decarboxylase by a new method for efficient markerless gene deletions in Gluconobacter oxydans

Gluconobacter oxydans, a biotechnologically relevant species which incompletely oxidizes a large variety of carbohydrates, alcohols, and related compounds, contains a gene for pyruvate decarboxylase (PDC). This enzyme is found only in very few species of bacteria where it is normally involved in anaerobic ethanol formation via acetaldehyde. In order to clarify the role of PDC in the strictly oxidative metabolism of acetic acid bacteria, we developed a markerless in-frame deletion system for strain G. oxydans 621H which uses 5-fluorouracil together with a plasmid-encoded uracil phosphoribosyltransferase as counter selection method and used this technique to delete the PDC gene (GOX1081) of G. oxydans 621H. The PDC deletion mutant accumulated large amounts of pyruvate but almost no acetate during growth on d-mannitol, d-fructose or in the presence of l-lactate. This suggested that in G. oxydans acetate formation occurs by decarboxylation of pyruvate and subsequent oxidation of acetaldehyde to acetate. This observation and the efficiency of the markerless deletion system were confirmed by constructing deletion mutants of two acetaldehyde dehydrogenases (GOX1122 and GOX2018) and of the acetyl-CoA-synthetase (GOX0412). Acetate formation during growth of these mutants on mannitol did not differ significantly from the wild-type strain.     

Effects of alcohol and solvent on the performance of lipase from Candida sp. in enantioselective esterification of racemic ibuprofen

The Candida sp. lipase prepared in our lab was used for the resolution of racemic ibuprofen. In order to study the effects of alcohol and solvent on the performance of Candida sp. lipase in enantioselective esterification of racemic ibuprofen, different alcohols were chosen as acyl acceptors in the same solvent, and identical substrates were used in different solvents. The reactions were performed under controlled water activity, thereby permitting the influences of the alcohols and the solvents to be separated from their ability to strip water from the solid enzyme. The results showed that alcohols and solvents had great effects on the performance of Candida sp. lipase.     

Synthesis of (E)-cinnamyl ester derivatives via a greener Steglich esterification

Cinnamic acid derivatives are known antifungal, antimicrobial, antioxidant, and anticancer compounds. We have developed a facile and mild methodology for the synthesis of (E)-cinnamate derivatives using a modified Steglich esterification of (E)-cinnamic acid. Using acetonitrile as the solvent, rather than the typical chlorinated solvent, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as the coupling agent enables ester conversion in 45?min with mild heating (40–45?°C) and an average yield of 70% without need for further purification. These conditions were used to couple (E)-cinnamic acid with 1° and 2° aliphatic alcohols, benzylic and allylic alcohols, and phenols. This work demonstrates a facile and greener methodology for Steglich esterification reactions.     

The impact of ionic mercury on antioxidant defenses in two mercury-sensitive anaerobic bacteria

While the toxicological effects of mercury (Hg) are well studied in mammals, little is known about the mechanisms of toxicity to bacterial cells lacking an Hg resistance (mer) operon. We determined that Shewanella oneidensis MR-1 is more sensitive to ionic mercury [Hg(II)] under aerobic conditions than in fumarate reducing conditions, with minimum inhibitory concentrations of 0.25 and 2 μM respectively. This increased sensitivity in aerobic conditions is not due to increased import, as more Hg is associated with cellular material in fumarate reducing conditions than in aerobic conditions. In fumarate reducing conditions, glutathione may provide protection, as glutathione levels decrease in a dose-dependent manner, but this does not occur in aerobic conditions. Hg(II) does not change the redox state of thioredoxin in MR1 in either fumarate reducing conditions or aerobic conditions, although thioredoxin is oxidized in Geobacter sulfurreducens PCA in response to Hg(II) treatment. However, treatment with 0.5 μM Hg(II) increases lipid peroxidation in aerobic conditions but not in fumarate reducing conditions in MR-1. We conclude that the enhanced sensitivity of MR-1 to Hg(II) in aerobic conditions is not due to differences in intracellular responses, but due to damage at the cell envelope.     

Biphasic aqueous/organic biotransformation of acetaldehyde and benzaldehyde by Zymomonas mobilis pyruvate decarboxylase

Zymomonas mobilis pyruvate decarboxylase (PDC) transformed acetaldehyde and benzaldehyde into (R)-phenylacetylcarbinol (PAC), the precursor for the synthesis of ephedrine and pseudoephedrine. Organic solvents were screened for a biphasic biotransformation with the enzyme in an aqueous phase and the toxic substrates delivered through the organic phase. In the absence of substrates a second phase of 1-pentanol, hexadecane or MTBE (methyl tertiary-butyl ether) stabilized the PDC activity in comparison to a control without added solvent. Organic phase solvents for optimal PAC production had partitioning coefficient (log P) values between 0.8 and 2.8 (production of more than 8 mg PAC/ U PDC), however there was no correlation between enzyme stability and log P. Best PAC formation was observed with the eight tested alcohols, which in contrast to the other solvents allowed lower initial concentrations of toxic acetaldehyde (54-81 mM) in the aqueous phase. 1-pentanol, 1-hexanol, and isobutanol resulted in the highest specific PAC production of 11 mg PAC /U PDC. Without the addition of an organic phase, only 1.2 mg/U was formed.