scholarly journals Gold(I)-catalysed synthesis of a furan analogue of thiamine pyrophosphate

2014 ◽  
Vol 10 ◽  
pp. 2580-2585 ◽  
Author(s):  
Amjid Iqbal ◽  
El-Habib Sahraoui ◽  
Finian J Leeper
Keyword(s):  
Zymomonas Mobilis ◽  
Furan Ring ◽  
Pyruvate Decarboxylase ◽  
Thiamine Pyrophosphate ◽  
Reaction Intermediates ◽  
Strong Inhibitor ◽  
E Coli ◽  
His Tag ◽  
Efficient Route ◽  
Thiazolium Ring

An analogue of thiamine having a furan ring in place of the thiazolium ring has been synthesised by a short and efficient route, involving gold(I)-catalysed cyclisation of an alkynyl alcohol to form the furan ring. The furan analogue of thiamine diphosphate (ThDP) was also made and tested for binding to and inhibition of pyruvate decarboxylase (PDC) from Zymomonas mobilis (overexpressed in E. coli with a N-terminal His-tag). It is a very strong inhibitor, with a K i value of 32.5 pM. It was also shown that the furan analogue of thiamine can be functionalised at the C-2 position, which will allow access to mimics of reaction intermediates of various ThDP-dependent enzymes.

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 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.

scholarly journals Differential expression of Zymomonas mobilis sucrase genes (sacB and sacC) in Escherichia coli and sucrase mutants of Zymomonas mobilis

2004 ◽  
Vol 47 (3) ◽  
pp. 329-338 ◽  
Author(s):  
Sangiliyandi Gurunathan ◽  
Paramasamy Gunasekaran
Keyword(s):  
Copy Number ◽  
Zymomonas Mobilis ◽  
Northern Blot Analysis ◽  
Shuttle Vector ◽  
Transcript Levels ◽  
E Coli ◽  
Low Copy Number ◽  
Genes Encoding ◽  
Sacb Gene ◽  
In Cells

The sacB and sacC genes encoding levansucrase and extracellular sucrase respectively were independently subcloned in pBluescript (high copy number) and in Z. mobilis-E. coli shuttle vector, pZA22 (low copy number). The expression of these genes were compared under identical background of E. coli and Z. mobilis host. The level of sacB gene expression in E. coli was almost ten fold less than the expression of sacC gene, irrespective of the growth medium or the host strain. In Z. mobilis the expression of sacB and sacC genes was shown to be subject to carbon source dependent regulation. The transcript of sacB and sacC was three fold higher in cells grown on sucrose than in cells grown on glucose/fructose. Northern blot analysis revealed that the transcript levels of sacC was approximately 2-3 times higher than that of sacB. These results suggested that the expression of sacC gene was more pronounced than sacB.

scholarly journals High-Level Acetaldehyde Production in Lactococcus lactis by Metabolic Engineering

2005 ◽  
Vol 71 (2) ◽  
pp. 1109-1113 ◽  
Author(s):  
Roger S. Bongers ◽  
Marcel H. N. Hoefnagel ◽  
Michiel Kleerebezem
Keyword(s):  
Metabolic Engineering ◽  
Lactococcus Lactis ◽  
Zymomonas Mobilis ◽  
Nadh Oxidase ◽  
Pyruvate Decarboxylase ◽  
Resting Cells ◽  
Anaerobic Conditions ◽  
High Level ◽  
Acetaldehyde Production ◽  
Combined Overexpression

ABSTRACT Efficient conversion of glucose to acetaldehyde is achieved by nisin-controlled overexpression of Zymomonas mobilis pyruvate decarboxylase (pdc) and Lactococcus lactis NADH oxidase (nox) in L. lactis. In resting cells, almost 50% of the glucose consumed could be redirected towards acetaldehyde by combined overexpression of pdc and nox under anaerobic conditions.

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.

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