Biochemical and genetic characterization of the Enterococcus faecalis oxaloacetate decarboxylase complex.

Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn2+.

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO(2). They are activated by Mn(2+), a metal ion that coordinates to the protein through the epsilon-amino group of a lysine residue, the N(epsilon-2)-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the epsilon-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the epsilon-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn(2+) in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn(2+) affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn(2+). In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.

Post-translational processing, metabolic stability and catalytic efficiency of oat arginine decarboxylase expressed in Trypanosoma cruzi epimastigotes.

Trypanosoma cruzi epimastigotes are auxotrophic for polyamines because they are unable to synthesize putrescine de novo. This deficiency is due to the absence of ornithine and arginine decarboxylase genes in the parasite genome. We have been able to obtain transgenic T. cruzi expressing heterologous genes coding for these enzymes. Since arginine decarboxylase normal expression in oat requires a post-translational proteolytic cleavage of an enzyme precursor, we have investigated whether a similar processing occurs inside the transformed protozoa expressing oat arginine decarboxylase or the same enzyme attached to a C-terminal (his)(6)-tag. We were able to demonstrate that the post-translational processing also takes place inside the transgenic parasites. This cleavage is probably the result of a general proteolytic activity of T. cruzi acting on a protease-sensitive region of the protein. Interestingly, the (his)(6)-tagged enzyme expressed in the transformed parasites showed considerably increased metabolic stability and catalytic efficiency.

Purification and characterization of a p-coumarate decarboxylase and a vinylphenol reductase from Brettanomyces bruxellensis.

The presence of Brettanomyces bruxellensis has been correlated with an increase of phenolic aromas in wine. The production of these aromas results from the metabolization of cinnamic acids, present in the wine, to their ethyl derivatives. Hence, the participation of two enzymes has been proposed: a p-coumarate decarboxylase (CD) and a vinylphenol reductase (VR). Both enzymes were purified and characterized from B. bruxellensis. In denaturing conditions, the CD enzyme had a molecular mass of 21 kDa, while in native conditions its mass was 41 kDa. The optimal activity was obtained at a temperature of 40 degrees C and a pH of 6.0. For p-coumaric acid, the Km value and Vmax were 1.22+/-0.08 mM and 98+/-0.15 micromol/min mg, respectively. The VR enzyme had a molecular mass of 37 kDa in SDS-PAGE, while in natural conditions its mass was 118 kDa. The Km value was > 3.37+/-2.05 mM and its Vmax was 107.62+/-50.38 micromol/min mg for NADPH used as a cofactor. Both enzymatic activities were stable at pH 3.4, but in the presence of ethanol the CD activity decreased drastically while the VR activity was more stable. This is the first report that shows the presence of a CD and a VR enzyme in B. bruxellensis.

Leishmania mexicana amazonensis: development of a peptide tag useful for labeling and purifying biotinylated recombinant proteins.

A number of peptide tags are available to facilitate the characterization of recombinant proteins. We have tested the bacterial oxaloacetate decarboxylase biotinylation domain for its efficacy in tagging recombinant proteins in vivo in Leishmania. To achieve efficient biotinylation, Leishmania also had to be co-transformed with the gene for bacterial biotin protein ligase (birA gene product). The recombinant chimeric protein could be detected on blots probed with avidin-horseradish peroxidase and purified on immobilized monomeric avidin resins.

[Experimental porphyria induced by chlorinated hydrocarbons. Studies of porphyrinogen carboxy-lyase in the experimental model of human cutaneous delayed porphyria]. / Porfiria experimental por hidrocarburos clorados. Estudios sobre la porfirinogeno carboxi-liasa en el, modelo experimental de la porfiria cutanea tarda humana.

The present work tries to elucidate if the strong decrease of porphyrinogen carboxy-lyase (PCL) observed in the experimental porphyria caused by hexachlorobenzene (HCB) is due to the presence of an inhibitor or to a modification of the protein structure of the enzyme. For this purpose: a) cross-assays and heating ones were performed in order to look for the existence of a compound, that present in porphyric animals, would be responsible for the decrease of PCL activity found in them; b) the effect in vitro of HCB, HCB metabolites and other related compounds was studied to find the inhibitor of PCL activity and to look for a relation structure-inhibitory effect; c) hepatic PCL from porphyric and normal rats was purified, and enzyme properties were comparatively studied looking for structural differences between the enzymes obtained from both animal lots. The results indicate that: a) the heat deproteinized porphyric liver preparation produces an inhibition on the normal preparation, but it is smaller than the decrease produced in vivo by the HCB on this enzyme activity, thus other reasons may explain this behavior of PCL in intoxicated animals; b) the HCB had no effect on PCL activity, the phenolic compounds exhibited inhibitions of variable extent that were increased by the presence of electrophilic substituents on the benzenic ring. Pentachlorophenol, the main HCB metabolite, produced inhibition in the in vitro assays, but at doses that were not physiologically significant; thus, it seems not to be the inhibitor found in the heating assays; c) purification of 110 times for the hepatic PCL of both porphyric and normal rats was obtained. Incubation conditions, the effect of salts and chelating agents, the chromatographic behavior in DEAE-cellulose and Sephadex G-100 columns, were comparatively studied with both enzymatic preparations. The effect of sodium diethyldithiocarbamate, sodium pyrophosphate, dithiothreitol, temperature, pH and O2, as well as the chromatographic behavior, would suggest that structural differences in the PCL of porphyric animals may exist; the presence of a thermostable inhibitor could also contribute to the decrease of PCL activity due to HCB.

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis.

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K(m) 0.042mm), 2.0mm-ATP (K(m) 0.19mm) and 8mm-Mg(2+) at 40 degrees C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5-6.5 with 0.1mm-5-pyrophosphomevalonate (K(m) 0.004mm), 1.5mm-ATP (K(m) 0.12mm) and 2mm-Mg(2+). The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.