scholarly journals Pyruvate decarboxylase from Zymomonas mobilis. Structure and re-activation of apoenzyme by the cofactors thiamin diphosphate and magnesium ion

1991 ◽  
Vol 276 (2) ◽  
pp. 439-445 ◽  
Author(s):  
R J Diefenbach ◽  
R G Duggleby
Keyword(s):  
Zymomonas Mobilis ◽  
Dipicolinic Acid ◽  
Gel Filtration ◽  
Pyruvate Decarboxylase ◽  
Tryptophan Fluorescence ◽  
Subunit Structure ◽  
Thiamin Diphosphate ◽  
Reversible Binding ◽  
Cofactor Binding ◽  
Series Of Experiments

To study the mechanism of re-activation of Zymomonas mobilis pyruvate decarboxylase apoenzyme by its cofactors thiamin diphosphate and Mg2+, cofactor-free enzyme was prepared by dialysis against 1 mM-dipicolinic acid at pH 8.2. This apoenzyme was then used in a series of experiments that included determination of: (a) the affinity towards one cofactor when the other was present at saturating concentrations; (b) cofactor-binding rates by measuring the quenching of tryptophan fluorescence on the apoenzyme; (c) the effect of replacement of cofactors with various analogues; (d) the stoichiometry of bound cofactors in holoenzyme; and (e) the molecular mass of apoenzyme by gel filtration. The results of these experiments form the basis for a proposed model for the re-activation of Z. mobilis pyruvate decarboxylase apoenzyme by its cofactors. In this model there exists two alterative but equivalent pathways for cofactor binding. In each pathway the first step is an independent reversible binding of either thiamin diphosphate (Kd 187 microM) or Mg2+ (Kd 1.31 mM) to free apoenzyme. When both cofactors are present, the second cofactor-binding step to form active holoenzyme is a slow quasi-irreversible step. This second binding step is a co-operative process for both thiamin diphosphate (Kd 0.353 microM) and Mg2+ (Kd 2.47 microM). Both the apo- and the holo-enzyme have a tetrameric subunit structure, with cofactors binding in a 1:1 ratio with each subunit.

scholarly journals Aspartate-27 and glutamate-473 are involved in catalysis by Zymomonas mobilis pyruvate decarboxylase

1999 ◽  
Vol 339 (2) ◽  
pp. 255-260 ◽  
Author(s):  
Alan K. CHANG ◽  
Peter F. NIXON ◽  
Ronald G. DUGGLEBY
Keyword(s):  
Zymomonas Mobilis ◽  
Specific Activity ◽  
Pyruvate Decarboxylase ◽  
Quantitative Model ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Thiamin Diphosphate ◽  
Cofactor Binding ◽  
A Minor ◽  
Mutant Forms

Zymomonas mobilis pyruvate decarboxylase (EC 4.1.1.1) was subjected to site-directed mutagenesis at two acidic residues near the thiamin diphosphate cofactor in the active site. Asp-27 was changed to Glu or Asn, and Glu-473 was mutated to Asp (E473D) or Gln (E473Q). Each mutant protein was purified to near-homogeneity, and the kinetic and cofactor-binding properties were compared with those of the wild-type protein. Despite the very conservative nature of these alterations, all mutants had a very low, but measurable, specific activity ranging from 0.025% (E473Q) to 0.173% (E473D) of the wild type. With the exception of E473Q, the mutants showed small decreases in the affinity for thiamin diphosphate, and binding of the second cofactor (Mg2+) was also weakened somewhat. With E473Q, both cofactors seemed to be very tightly bound so that they were not removed by the treatment that was effective for the wild-type enzyme and other mutant forms. All mutants showed minor changes in the Km for substrate, but these alterations did not account for the low activities. These low specific activities, accompanied by little change in the Km for pyruvate, are consistent with a quantitative model of the catalytic cycle in which the main effect of the mutations is to slow the decarboxylation step with a minor change in the rate constant for pyruvate binding.

scholarly journals Investigation of the cofactor-binding site of Zymomonas mobilis pyruvate decarboxylase by site-directed mutagenesis

1994 ◽  
Vol 300 (1) ◽  
pp. 7-13 ◽  
Author(s):  
J M Candy ◽  
R G Duggleby
Keyword(s):  
Zymomonas Mobilis ◽  
Pyruvate Decarboxylase ◽  
Active Enzyme ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Kinetic Properties ◽  
Binding Motif ◽  
Sequence Motif ◽  
Cofactor Binding

Several enzymes require thiamin 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 and its simple kinetic properties. A sequence motif of approx. 30 residues, beginning with a glycine-aspartate-glycine (-GDG-) triplet and ending with a double asparagine (-NN-) sequence, has been identified in many of these enzymes [Hawkins, Borges and Perham (1989) FEBS Lett. 255, 77-82]. Other residues within this putative ThDP-binding motif are conserved, but to a lesser extent, including a glutamate and a proline residue. The role of the elements of this motif has been clarified by the determination of the three-dimensional structure of three of these enzymes [Muller, Lindqvist, Furey, Schulz, Jordan and Schneider (1993) Structure 1, 95-103]. Four of the residues within this motif were modified by site-directed mutagenesis of the cloned PDC gene to evaluate their role in cofactor binding. The mutant proteins were expressed in Escherichia coli and found to purify normally, indicating that the tertiary structure of these enzymes had not been grossly perturbed by the amino acid substitutions. We have shown previously [Diefenbach, Candy, Mattick and Duggleby (1992) FEBS Lett. 296, 95-98] that changing the aspartate in the -GDG- sequence to glycine, threonine or asparagine yields an inactive enzyme that is unable to bind ThDP, therefore verifying the role of the ThDP-binding motif. Here we demonstrate that substitution with glutamate yields an active enzyme with a greatly reduced affinity for both ThDP and Mg2+, but with normal kinetics for pyruvate. Unlike the wild-type tetrameric enzyme, this mutant protein usually exists as a dimer. Replacement of the second asparagine of the -NN- sequence by glutamine also yields an inactive enzyme which is unable to bind ThDP, whereas replacement with an aspartate residue results in an active enzyme with a reduced affinity for ThDP but which displays normal kinetics for both Mg2+ and pyruvate. Replacing the conserved glutamate with aspartate did not alter the properties of the enzyme, while the conserved proline, thought to be required for structural reasons, could be substituted with glycine or alanine without inactivating the enzyme, but these changes did reduce its stability.

scholarly journals The role of residues glutamate-50 and phenylalanine-496 in Zymomonas mobilis pyruvate decarboxylase

1996 ◽  
Vol 315 (3) ◽  
pp. 745-751 ◽  
Author(s):  
Judith M. CANDY ◽  
Jinichiro KOGA ◽  
Peter F. NIXON ◽  
Ronald G. DUGGLEBY
Keyword(s):  
Fluorescence Quenching ◽  
Zymomonas Mobilis ◽  
Specific Interaction ◽  
Pyruvate Decarboxylase ◽  
Site Directed Mutagenesis ◽  
Kinetic Properties ◽  
Subunit Structure ◽  
Wild Type ◽  
Cloned Gene

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.

scholarly journals Recombinant production ofZymomonas mobilispyruvate decarboxylase in the haloarchaeonHaloferax volcanii

Archaea ◽  
2005 ◽  
Vol 1 (5) ◽  
pp. 327-334 ◽  
Author(s):  
Steven J. Kaczowka ◽  
Christopher J. Reuter ◽  
Lee A. Talarico ◽  
Julie A. Maupin-Furlow
Keyword(s):  
Stationary Phase ◽  
Zymomonas Mobilis ◽  
Gel Filtration ◽  
Pyruvate Decarboxylase ◽  
Gram Negative Bacteria ◽  
Late Stationary Phase ◽  
E Coli ◽  
Recombinant Production ◽  
Physiological Properties ◽  
Specific Mrna

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 ofZymomonas mobilis(Zmpdc) was expressed inHaloferax volcanii. This gene has been used successfully to channel pyruvate to ethanol in various Gram-negative bacteria, includingEscherichia coli. Although the ionic strength of theH. volcaniicytosol differs over 15-fold from that ofE. coli, gel filtration and circular dichroism revealed no difference in secondary structure between the ZmPDC protein isolated from either of these hosts. Like theE. colipurified enzyme, ZmPDC fromH. volcaniicatalyzed the nonoxidative decarboxylation of pyruvate. A decrease in the amount of soluble ZmPDC protein was detected asH. volcaniitransitioned from log phase to late stationary phase that was inversely proportional to the amount ofpdc-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.

scholarly journals Preliminary crystallographic data for the thiamin diphosphate-dependent enzyme pyruvate decarboxylase from brewers' yeast.

1990 ◽  
Vol 265 (29) ◽  
pp. 17413-17415
Author(s):  
F Dyda ◽  
W Furey ◽  
S Swaminathan ◽  
M Sax ◽  
B Farrenkopf ◽  
...  
Keyword(s):  
Pyruvate Decarboxylase ◽  
Crystallographic Data ◽  
Thiamin Diphosphate ◽  
Brewers Yeast

scholarly journals Purification and subunit structure of argininosuccinate lyase from Chlamydomonas reinhardi

1977 ◽  
Vol 167 (1) ◽  
pp. 71-75 ◽  
Author(s):  
R F Matagne ◽  
J P Schlösser
Keyword(s):  
Enzyme Activity ◽  
Crude Extract ◽  
Enzyme Preparation ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Activity Analysis ◽  
Disc Electrophoresis ◽  
Subunit Structure ◽  
Argininosuccinate Lyase

Argininosuccinate lyase (EC 4.3.2.1) was purified by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose and gel filtration on Sephadex G-200. The final enzyme preparation was purified 46-fold compared with the crude extract. Electrophoresis of this preparation revealed three bands, the major one having the enzyme activity. Analysis of the enzyme by gel filtration and by disc electrophoresis (in two different concentrations of acrylamide) gave mol.wts. of 200000 (+/- 15000) and 190000 (+/- 20000) respectively. Treatment with sodium dodecyl sulphate and mercaptoethanol dissociated the enzyme into subunits of mol.wt. 39000 (+/-2000). The results are indicative of the multimeric structure of the enzyme, which is composed of five (perhaps four or six) identical subunits.

scholarly journals Ethanologenesis and respiration in a pyruvate decarboxylase-deficient Zymomonas mobilis

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Reinis Rutkis ◽  
Inese Strazdina ◽  
Zane Lasa ◽  
Per Bruheim ◽  
Uldis Kalnenieks
Keyword(s):  
Parent Strain ◽  
Zymomonas Mobilis ◽  
Nadh Oxidase ◽  
Pyruvate Decarboxylase ◽  
Production Ratio ◽  
Pyruvate Node ◽  
Branching Point ◽  
Adh Activity ◽  
Ethanol Synthesis

Abstract Objective Zymomonas mobilis is an alpha-proteobacterium with a rapid ethanologenic pathway, involving Entner–Doudoroff (E–D) glycolysis, pyruvate decarboxylase (Pdc) and two alcohol dehydrogenase (ADH) isoenzymes. Pyruvate is the end-product of the E–D pathway and the substrate for Pdc. Construction and study of Pdc-deficient strains is of key importance for Z. mobilis metabolic engineering, because the pyruvate node represents the central branching point, most novel pathways divert from ethanol synthesis. In the present work, we examined the aerobic metabolism of a strain with partly inactivated Pdc. Results Relative to its parent strain the mutant produced more pyruvate. Yet, it also yielded more acetaldehyde, the product of the Pdc reaction and the substrate for ADH, although the bulk ADH activity was similar in both strains, while the Pdc activity in the mutant was reduced by half. Simulations with the kinetic model of Z. mobilis E-D pathway indicated that, for the observed acetaldehyde to ethanol production ratio in the mutant, the ratio between its respiratory NADH oxidase and ADH activities should be significantly higher, than the measured values. Implications of this finding for the directionality of the ADH isoenzyme operation in vivo and interactions between ADH and Pdc are discussed.

Sign in / Sign up

Export Citation Format

Share Document