Dataset Information

Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii.

ABSTRACT: The unusual physiological properties of archaea (e.g., growth in extreme salt concentration, temperature and pH) make them ideal platforms for metabolic engineering. Towards the ultimate goal of modifying an archaeon to produce bioethanol or other useful products, the pyruvate decarboxylase gene of Zymomonas mobilis (Zm pdc) was expressed in Haloferax volcanii. This gene has been used successfully to channel pyruvate to ethanol in various Gram-negative bacteria, including Escherichia coli. Although the ionic strength of the H. volcanii cytosol differs over 15-fold from that of E. coli, gel filtration and circular dichroism revealed no difference in secondary structure between the ZmPDC protein isolated from either of these hosts. Like the E. coli purified enzyme, ZmPDC from H. volcanii catalyzed the nonoxidative decarboxylation of pyruvate. A decrease in the amount of soluble ZmPDC protein was detected as H. volcanii transitioned from log phase to late stationary phase that was inversely proportional to the amount of pdc-specific mRNA. Based on these results, proteins from non-halophilic organisms can be actively synthesized in haloarchaea; however, post-transcriptional mechanisms present in stationary phase appear to limit the amount of recombinant protein expressed.

SUBMITTER: Kaczowka SJ

PROVIDER: S-EPMC2685553 | biostudies-literature | 2005 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii.

Kaczowka Steven J SJ Reuter Christopher J CJ Talarico Lee A LA Maupin-Furlow Julie A JA

Archaea (Vancouver, B.C.) 20050501 5

The unusual physiological properties of archaea (e.g., growth in extreme salt concentration, temperature and pH) make them ideal platforms for metabolic engineering. Towards the ultimate goal of modifying an archaeon to produce bioethanol or other useful products, the pyruvate decarboxylase gene of Zymomonas mobilis (Zm pdc) was expressed in Haloferax volcanii. This gene has been used successfully to channel pyruvate to ethanol in various Gram-negative bacteria, including Escherichia coli. Altho ...[more]

PMID: 15876566

Similar Datasets

Project description:Haloferax volcanii is a facultative anaerobic haloarchaeon that can grow using nitrate or dimethyl sulfoxide (DMSO) as a respiratory substrate under anaerobic conditions. Comparative transcriptome analysis of denitrifying and aerobic cells of H. volcanii indicated extensive changes in gene expression involving the activation of denitrification, suppression of DMSO respiration, and conversion of the heme biosynthetic pathway under denitrifying conditions. The anaerobic growth of H. volcanii by DMSO respiration was inhibited at nitrate concentrations of <1 mM, whereas nitrate-responsive growth inhibition was not observed in the ΔnarO mutant. A reporter assay demonstrated that the transcription of the dms operon was suppressed by nitrate. In contrast, the anaerobic growth of the ΔdmsR mutant by denitrification was little affected by the addition of DMSO. NarO has been identified as an activator of denitrification-related genes in response to anaerobic conditions, and here, we found that NarO is also involved in nitrate-responsive suppression of the dms operon. Nitrate-responsive suppression of DMSO respiration is known in several bacteria such as Escherichia coli and photosynthetic Rhodobacter species. This is the first report to show that a regulatory mechanism that suppresses DMSO respiration in response to nitrate exists not only in bacteria but also in haloarchaea. IMPORTANCE Haloferax volcanii can grow anaerobically by denitrification (nitrate respiration) or DMSO respiration. In facultative anaerobic bacteria that can grow by both nitrate respiration and DMSO respiration, nitrate respiration is preferentially induced when both nitrate and DMSO are available as the respiratory substrates. The results of transcriptome analysis, growth phenotyping, and reporter assays indicated that DMSO respiration is suppressed in response to nitrate in H. volcanii. The haloarchaeon-specific regulator NarO, which activates denitrification under anaerobic conditions, is suggested to be involved in the nitrate-responsive suppression of DMSO respiration.

Project description:Several enzymes require thiamine diphosphate (ThDP) as an essential cofactor, and we have used one of these, pyruvate decarboxylase (PDC; EC 4.1.1.1) from Zymomonas mobilis, as a model for this group of enzymes. It is well suited for this purpose because of its stability, ease of purification, homotetrameric subunit structure and simple kinetic properties. Crystallographic analyses of three ThDP-dependent enzymes [Müller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103] have suggested that an invariant glutamate participates in catalysis. In order to evaluate the role of this residue, identified in PDC from Zymomonas mobilis as Glu-50, it has been altered to glutamine and aspartate by site-directed mutagenesis of the cloned gene. The mutant proteins were expressed in Escherichia coli. Here we demonstrate that substitution with aspartate yields an enzyme with 3% of the activity of the wild-type, but with normal kinetics for pyruvate. Replacement of Glu-50 with glutamine yields an enzyme with only 0.5% of the catalytic activity of the wild-type enzyme. Each of these mutant enzymes has a decreased affinity for both ThDP and Mg2+. It has been reported that the binding of cofactors to apoPDC quenches the intrinsic tryptophan fluorescence [Diefenbach and Duggleby (1991) Biochem. J. 276, 439-445] and we have identified the residue responsible as Trp-487 [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98]. Although this residue is some distance from the cofactor binding site, it lies in the dimer interface, and the proposal has been put forward [Dyda, Furey, Swaminathan, Sax, Farrenkopf and Jordan (1993) Biochemistry 32, 6165-6170] that alteration of ring stacking with Phe-496 of the adjacent subunit is the mechanism of fluorescence quenching when cofactors bind. The closely related enzyme indolepyruvate decarboxylase (from Enterobacter cloacae) has a leucine residue at the position corresponding to Phe-496 but shows fluorescence quenching properties that are similar to those of PDC. This suggests that the fluorescence quenching is due to some perturbation of the local environment of Trp-487 rather than to a specific interaction with Phe-496. This latter hypothesis is supported by our data: mutation of this phenylalanine to leucine, isoleucine or histidine in PDC does not eliminate the fluorescence quenching upon addition of cofactors.

Dataset Information

Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii.

Publications

Recombinant production of Zymomonas mobilis pyruvate decarboxylase in the haloarchaeon Haloferax volcanii.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets