Escherichia coli Malic Enzymes: Two Isoforms with Substantial Differences in Kinetic Properties, Metabolic Regulation, and Structure (original) (raw)
Related papers
Characterization of an oxaloacetate decarboxylase that belongs to the malic enzyme family
FEBS letters, 2004
The citM gene from Lactococcus lactis CRL264 was demonstrated to encode for an oxaloacetate decarboxylase. The enzyme exhibits high levels of similarity to malic enzymes (MEs) from other organisms. CitM was expressed in Escherichia coli, purified and its oxaloacetate decarboxylase activity was demonstrated by biochemical and genetic studies. The highest oxaloacetate decarboxylation activity was found at low pH in the presence of manganese, and the Km value for oxaloacetate was 0.52+/-0.03 mM. However, no malic activity was found for this enzyme. Our studies clearly show a new group of oxaloacetate decarboxylases associated with the citrate fermentation pathway in gram-positive bacteria. Furthermore, the essential catalytic residues were found to be conserved in all members of the ME family, suggesting a common mechanism for oxaloacetate decarboxylation.
Journal of Biological Chemistry, 1998
Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate in conjunction with the reduction of a nicotinamide cofactor. We determined the DNA sequence and transcriptional start sites of the genes encoding the diphosphopyridine nucleotide-dependent malic enzyme (DME, EC 1.1.1.39) and the triphosphopyridine nucleotide-dependent malic enzyme (TME, EC 1.1.1.40) of Rhizobium (Sinorhizobium) meliloti. The predicted DME and TME proteins contain 770 and 764 amino acids, respectively, and are approximately 320 amino acids larger than previously characterized prokaryotic malic enzymes. The increased size of DME and TME resides in the C-terminal extensions which are similar in sequence to phosphotransacetylase enzymes (EC 2.3.1.8). Modified DME and TME proteins which lack this C-terminal region retain malic enzyme activity but are unable to oligomerize into the native state. Data base searches have revealed that similar chimeric malic enzymes were uniquely present in Gram-negative bacteria. Thus DME and TME appear to be members of a new class of malic enzyme characterized by the presence of a phosphotransacetylase-like domain at the C terminus of the protein.
Engineering of Escherichia coli for Krebs cycle-dependent production of malic acid
Microbial cell factories, 2018
Malate is a C4-dicarboxylic acid widely used as an acidulant in the food and beverage industry. Rational engineering has been performed in the past for the development of microbial strains capable of efficient production of this metabolite. However, as malate can be a precursor for specialty chemicals, such as 2,4-dihydroxybutyric acid, that require additional cofactors NADP(H) and ATP, we set out to reengineer Escherichia coli for Krebs cycle-dependent production of malic acid that can satisfy these requirements. We found that significant malate production required at least simultaneous deletion of all malic enzymes and dehydrogenases, and concomitant expression of a malate-insensitive PEP carboxylase. Metabolic flux analysis using C-labeled glucose indicated that malate-producing strains had a very high flux over the glyoxylate shunt with almost no flux passing through the isocitrate dehydrogenase reaction. The highest malate yield of 0.82 mol/mol was obtained with E. coli Δmdh Δm...
2013
The malate-pyruvate conversion pathway is catalyzed by two malic enzyme isomers, MaeA and MaeB. qRT-PCR was carried out under malate and pyruvate supplemented conditions to understand the dynamics of maeA and maeB gene expression. maeA expression was elevated by malate, and maeB expression was inhibited by levels of both malate and pyruvate higher than 0.1 mM. Green fluorescent protein (GFP) reporter plasmids were also constructed by integration of the maeA/maeB promoter with the gfp gene. We showed that maeA driven GFP expression was positively and negatively correlated with extracellular malate and pyruvate induction. In contrast, no significant changes in maeB driven GFP expression were observed under both malate and pyruvate supplemented conditions.
Properties of Malic Enzyme from the Aerobic Methanotroph Methylosinus trichosporium
Biokhimiya, 2019
Recombinant malic enzyme from the aerobic methanotroph Methylosinus trichosporium was obtained by hetero logous expression in Escherichia coli and purified by affinity metal chelating chromatography. The homohexameric enzyme of 6×80 kDa catalyzed the reversible reaction of oxidative decarboxylation of malate to pyruvate in the presence of mono and divalent cations and NADP + as a cofactor. The k cat /K m ratio indicated much higher catalytic efficiency of the malate decarboxylation reaction as compared with the pyruvate carboxylation reaction. Analysis of the protein sequence revealed that the C region of the enzyme contains a large domain homologous to phosphoacetyltransferase, but no phosphoacetyl transferase activity was detected either for a full chimeric malic enzyme or for the C end fragment obtained as a separate protein. This C end domain promoted activity of the malic enzyme.
Journal of Bacteriology, 2006
The Bacillus subtilis genome contains several sets of paralogs. An extreme case is the four putative malic enzyme genes maeA, malS, ytsJ, and mleA. maeA was demonstrated to encode malic enzyme activity, to be inducible by malate, but also to be dispensable for growth on malate. We report systematic experiments to test whether these four genes ensure backup or cover different functions. Analysis of single-and multiple-mutant strains demonstrated that ytsJ has a major physiological role in malate utilization for which none of the other three genes could compensate. In contrast, maeA, malS, and mleA had distinct roles in malate utilization for which they could compensate one another. The four proteins exhibited malic enzyme activity; MalS, MleA, and MaeA exhibited 4-to 90-fold higher activities with NAD ؉ than with NADP ؉ . YtsJ activity, in contrast, was 70-fold higher with NADP ؉ than with NAD ؉ , with K m values of 0.055 and 2.8 mM, respectively. lacZ fusions revealed strong transcription of ytsJ, twofold higher in malate than in glucose medium, but weak transcription of malS and mleA. In contrast, mleA was strongly transcribed in complex medium. Metabolic flux analysis confirmed the major role of YtsJ in malate-topyruvate interconversion. While overexpression of the NADP-dependent Escherichia coli malic enzyme MaeB did not suppress the growth defect of a ytsJ mutant on malate, overexpression of the transhydrogenase UdhA from E. coli partially suppressed it. These results suggest an additional physiological role of YtsJ beyond that of malate-to-pyruvate conversion.
A Catalytic Triad Is Responsible for Acid−Base Chemistry in the Ascaris suum NAD−Malic Enzyme
Biochemistry, 2005
The pH dependence of kinetic parameters of several active site mutants of the Ascaris suum NAD-malic enzyme was investigated to determine the role of amino acid residues likely involved in catalysis on the basis of three-dimensional structures of malic enzyme. Lysine 199 is positioned to act as the general base that accepts a proton from the 2-hydroxyl of malate during the hydride transfer step. The pH dependence of V/K malate for the K199R mutant enzyme reveals a pK of 5.3 for an enzymatic group required to be unprotonated for activity and a second pK of 6.3 that leads to a 10-fold loss in activity above the pK of 6.3 to a new constant value up to pH 10. The V profile for K199R is pH independent from pH 5.5 to pH 10 and decreases below a pK of 4.9. Tyrosine 126 is positioned to act as the general acid that donates a proton to the enolpyruvate intermediate to form pyruvate. The pH dependence of V/K malate for the Y126F mutant is qualitatively similar to K199R, with a requirement for a group to be unprotonated for activity with a pK of 5.6 and a partial activity loss of about 3-fold above a pK of 6.7 to a new constant value. The Y126F mutant enzyme is about 60000-fold less active than the wild-type enzyme. In contrast to K199R, the V rate profile for Y126F also shows a partial activity loss above pH 6.6. The wild-type pH profiles were reinvestigated in light of the discovery of the partial activity change for the mutant enzymes. The wild-type V/K malate pH-rate profile exhibits the requirement for a group to be unprotonated for catalysis with a pK of 5.6 and also shows the partial activity loss above a pK of 6.4. The wild-type V pH-rate profile decreases below a pK of 5.2 and is pH independent from pH 5.5 to pH 10. Aspartate 294 is within hydrogen-bonding distance to K199 in the open and closed forms of malic enzyme. D294A is about 13000-fold less active than the wild-type enzyme, and the pH-rate profile for V/K malate indicates the mutant is only active above pH 9. The data suggest that the pK present at about pH 5.6 in all of the pH profiles represents D294, and during catalysis D294 accepts a proton from K199 to allow K199 to act as a general base in the reaction. The pK for the general acid in the reaction is not observed, consistent with rapid tautomerization of enolpyruvate. No other ionizable group in the active site is likely responsible for the partial activity change observed in the pH profiles, and thus the group responsible is probably remote from the active site and the effect on activity is transmitted through the protein by a conformational change.