Escherichia coli malic enzymes: two isoforms with substantial differences in kinetic properties, metabolic regulation, and structure

Abstract

Malic enzymes (MEs) catalyze the oxidative decarboxylation of malate in the presence of a divalent metal ion. In eukaryotes, well-conserved cytoplasmic, mitochondrial, and plastidic MEs have been characterized. On the other hand, distinct groups can be detected among prokaryotic MEs, which are more diverse in structure and less well characterized than their eukaryotic counterparts. In Escherichia coli, two genes with a high degree of homology to ME can be detected: sfcA and maeB. MaeB possesses a multimodular structure: the N-terminal extension shows homology to ME, while the C-terminal extension shows homology to phosphotransacetylases (PTAs). In the present work, a detailed characterization of the products of E. coli sfcA and maeB was performed. The results indicate that the two MEs exhibit relevant kinetic, regulatory, and structural differences. SfcA is a NAD(P) ME, while MaeB is a NADP-specific ME highly regulated by key metabolites. Characterization of truncated versions of MaeB indicated that the PTA domain is not essential for the ME reaction. Nevertheless, truncated MaeB without the PTA domain loses most of its metabolic ME modulation and its native oligomeric state. Thus, the association of the two structural domains in MaeB seems to facilitate metabolic control of the enzyme. Although the PTA domain in MaeB is highly similar to the domains of proteins with PTA activity, MaeB and its PTA domain do not exhibit PTA activity. Determination of the distinct properties of recombinant products of sfcA and maeB performed in the present work will help to clarify the roles of MEs in prokaryotic metabolism.

FIG. 1.

(A) ME reaction. ME catalyzes the oxidative decarboxylation of the C₄ dicarboxylic acid malate to the C₃ acid pyruvate and CO₂. Depending on the type of ME, NAD or NADP is used. A metal ion cofactor is essential for the reaction. (B) The PEP-pyruvate-OAA node and its connection with glycolysis/gluconeogenesis and acetate activation. ME (SfcA and/or MaeB) in combination with PEP synthetase (PpsA) allows the formation of PEP, the direct precursor for gluconeogenesis, from TCA intermediates. Alternatively, PCK (PckA) can also direct C₄ intermediates to PEP. The conversion of PEP to pyruvate, an essential step in glycolysis, is catalyzed by pyruvate kinase (PykA or PykF). Also shown is the activation of acetate, which involves two possible pathways. One is catalyzed by the AMP-forming acetyl-CoA synthetase (ACS), and an alternative pathway uses acetate kinase (AcKA) in combination with PTA.

FIG. 2.

Recombinant E. coli MEs characterized in the present work. The scale indicates the number of amino acid residues in each protein. In boxes, the putative conserved domains (CDD) in SfcA and MaeB are indicated above each protein: ME, N-terminal domain; ME, NAD-binding domain and PTA domain. The truncated MaeB proteins Sp1, Sp2, and Sp3 (413, 388, and 468 amino acids, respectively) have the two ME domains, while MaeB-PTA (351 amino acids from the carboxyl-terminal part of MaeB) is composed of only the PTA domain.

FIG. 3.

Characterization of purified recombinant E. coli MEs and deletions. (A) Coomassie-stained SDS-PAGE (5 μg of each protein) of recombinant purified MaeB, SfcA, Sp1 (in this case, a total extract of the insoluble fraction of induced BL21 cells transformed with pET-Sp1), Sp2, Sp3, and MaeB-PTA. (B) Western blot using antibodies against recombinant MaeB. The proteins shown in panel A were transferred for Western analysis. Molecular mass markers (MWM) were run on the left of each gel. (C) Nondenaturing activity gels. Purified SfcA (2 mU) was detected as NAD ME activity, while purified MaeB (2 mU), Sp2 (10 μg), Sp3 (2 mU), and MaeB-PTA (10 μg) were detected as NADP ME activity. Native molecular markers were loaded on the left of each gel and Coomassie blue stained. (D) Coomassie blue-stained native gel (5 μg of each protein) of purified recombinant SfcA, MaeB, Sp2, Sp3, and MaeB-PTA. Native molecular markers were loaded on the left.

FIG. 4.

Regulatory properties of recombinant SfcA, MaeB, and Sp3. ME activity was measured for each isoform at pH 7.5 in the presence of 2 mM of each effector indicated on the y axes, with the exception of CoA, acetyl-CoA, and palmitoyl-CoA (50 μM). The malate concentration was kept at approximately one-fifth of the K_m value for each isoform (0.1 mM for SfcA, 0.6 mM for MaeB, and 1 mM for Sp3). The results are presented as the percentages of activity in the presence of the effectors in relation to the activity measured in the absence of the metabolites. The assays were done at least in triplicate, and the error bars indicate deviations between the measurements. Significant inhibition is shown by dark-gray bars with hatching. Significant activation is shown by light-gray bars with cross-hatching.

FIG. 5.

Comparison of the amino acid sequences from E. coli MaeB, PTA, and EutD. The sequences for E. coli MaeB, PTA, and EutD were aligned using CLUSTAL W (version 1.82). The similarity among these three proteins extended over the approximately 350 carboxyl-terminal amino acids from MaeB and PTA and the complete sequence from EutD. Conserved regions among the three proteins are marked with boxes.