Novel conserved hydrolase domain in the CLCA family of alleged calcium-activated chloride channels.

Advanced protein structure prediction methods combined with structure modeling show that the mammalian proteins, described until now as calcium-activated chloride channels (CLCAs), appear in fact to be membrane anchored metal-dependent hydrolases, possibly proteases. A metallohydrolase structural domain was predicted, unexpectedly, in the CLCA sequences. The well-conserved active site in the modeled structure of this hydrolase domain allows the prediction of catalytic action similar to that of metalloproteases. A number of protein structure prediction methods suggest the overall fold of the N-terminal hydrolase domain to be most similar to that of zinc metalloproteases (zincins), notably matrixins. This is confirmed by analysis of the three-dimensional structure model of the predicted CLCA1 hydrolase domain built using the known structure of the MMP-11 catalytic domain. Fragments of CLCA1 corresponding to the modeled hydrolase domain were expressed in Escherichia coli, and the resulting proteins were readily refolded into monomeric soluble protein, indicating formation of stable independent domains. The homology model was used to predict putative substrate sequences. Homologs of mammalian CLCA genes were detected in the genomes of a vast array of multicellular animals: lower vertebrates, tunicates, insects, crustaceans, echinoderms, and flatworms. The hydrolase prediction is discussed in the context of published experimentally determined effects of CLCA proteins on chloride conductance. Altered proteolytic processing of full-length CLCA1 containing a mutation abolishing the predicted hydrolase activity is shown as initial experimental evidence for a role of the hydrolase domain in processing of mature full-length CLCA1. The hydrolase prediction together with the presented experimental data add to doubts about the function of CLCAs as chloride channels and strengthen the hypothesis of channel-activating and/or channel-accessory roles.

A heterologous reductase affects the redox balance of recombinant Saccharomyces cerevisiae.

Recombinant Saccharomyces cerevisiae harbouring the xylose reductase (XR) gene XYL1 from Pichia stipitis was grown in anoxic chemostat culture at two different dilution rates. At each dilution rate a transient experiment, encompassing a shift in the sugar content of the medium from glucose to glucose plus xylose was performed. The steady states at the beginning and the end of the transients were compared in terms of specific product fluxes from glucose metabolism. At both dilution rates, the specific glycerol flux decreased and the specific acetate and CO2 fluxes increased. The specific ethanol flux was not affected. At the lower dilution rate, the production of biomass decreased during the transient, but at the higher dilution rate it increased. The changes in product pattern can be explained as being due to the redox perturbation caused by the consumption of reduced cofactors in the XR-catalysed reaction. Regeneration of NAD partly through xylose reduction instead of glycerol production decreased the formation of glycerol. Additionally, xylose reduction activated those pathways which produce reduced cofactors, such as acetate formation and the pentose phosphate pathway, indicated by increased acetate and CO2 production. The dual cofactor specificity of XR, with a preference for NADPH over NADH, was evident from the effects of xylose reduction on product fluxes. Comparison of the xylose reduction rates at low and high glucose flux indicated that the supply of reduced cofactors partly controlled the reaction rate. At the higher dilution rate, control by some other factor such as xylose transport or XR activity increased. Calculation of carbon balances at the steady states showed that all substrate carbon was recovered in biomass or products. Based on the specific product fluxes, calculations of quantitative cofactor balances at the steady states was attempted. However, sensitivity calculations showed that analysis errors in the range of 5% caused substantial errors in the cofactor balance, without affecting the carbon balance.

Fermentation strategies for improved heterologous expression of laccase in Pichia pastoris.

Improved expression of recombinant laccase by Pichia pastoris carrying the lcc1 cDNA isolated from Trametes versicolor was achieved by optimization of the cultivation conditions in a fermentor equipped with a methanol sensor system. The results indicated that the activity obtained in fermentor cultivations was at least 7 times higher than in shake-flask cultures. Three different strategies for fermentor cultivations were compared: A (30 degrees C, 1.0% methanol), B (20 degrees C, 1.0% methanol), and C (20 degrees C, 0.5% methanol). The laccase activity, particularly the specific activity, could be improved by decreasing the cultivation temperature. The mechanisms behind the temperature effect on the laccase activity may be ascribed to poor stability, release of more proteases from dead cells, and folding problems at higher temperature. The results showed that the methanol concentration had a marked effect on the production of active heterologous laccase. A fivefold higher volumetric laccase activity was obtained when the methanol concentration was kept at 0.5% instead of 1.0%. The detrimental effect of methanol on the production of recombinant laccase may be attributed to lower laccase stability, a higher proteolytic activity, and folding problems due to higher growth rate at 1.0% methanol.