Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology
ReviewThe pyruvate dehydrogenase multi-enzyme complex from Gram-negative bacteria
Introduction
2-Oxo acid dehydrogenase complexes convert 2-oxo acids to the corresponding acyl-CoA derivatives and produce NADH and CO2 in an irreversible reaction. Five members of this family are known at present, the pyruvate dehydrogenase complex (PDHC), the 2-oxoglutarate dehydrogenase complex (OGDHC), the branched-chain dehydrogenase complex (BCDHC), the glycine dehydrogenase complex (GDHC) and the acetoin dehydrogenase complex (ADHC). They function at strategic points in, usually aerobic, catabolic pathways and are therefore subject to stringent control. With the exception of GDHC these complexes share common building principles. Many peripheral components of the same type bind to a central core with octahedral (24 subunits) or icosahedral (60 subunits) symmetry. The molecular mass ranges from 0.7 to more than 10 MDa. Within this common architectural design many variations exist.
PDHC converts pyruvate to acetyl-CoA (Fig. 1) by the combined action of three enzymes, pyruvate dehydrogenase/decarboxylase (E1p), acetyltransferase (E2p) and dihydrolipoamide dehydrogenase (E3). The bacterial complexes are regulated by allosteric mechanisms and product inhibition. They lack regulatory proteins, such as pyruvate dehydrogenase phosphatase and kinase and the E3-binding protein (protein X), which are present in the eukaryotic complexes [1].
PDHC from Gram-negative bacteria possess an octahedral E2p core while the Gram-positive and eukaryotic PDHC complexes are based on an icosahedral core. The reader is referred to recent reviews for a comprehensive description of these and the other complexes [2, 3, 4]. In this review, we will restrict ourselves to the structure and enzymology of the PDHC from Gram-negative bacteria. Catalysis by each of the constituting enzymes is described on a structural basis if available, combined with mutagenesis of active site residues. Multi-enzyme catalysis is then discussed in terms of integration of these individual reactions by complex formation and active site coupling, provided by the ‘swinging arms’ of the substrate carrying lipoyl domains, bound to the core enzyme E2.
Section snippets
Gene organization of pyruvate dehydrogenase complexes from Gram-negative bacteria
At present, the genes encoding the PDHC from 8 Gram-negative bacteria have been characterized, in some cases partly. These are Escherichia coli [5, 6, 7, 8], Azotobacter vinelandii [9, 10, 11], Haemophilus influenzae (accession numbers: EMBL HI 32802 and HI 32803), Alcaligenes eutrophus [12], Neisseria meningitidis ([13], accession numbers: x82637, x77920), Neisseria gonorrhoeae (B.A. Roe, S. Clifton, D.W. Dyer, Genococcal Genome Sequencing Project), Pseudomonas aeruginosa [14] and Zymomonas
The pyruvate dehydrogenase/decarboxylase component (E1p)
The pyruvate dehydrogenase component (E1p) catalyses the reductive acetylation of the E2-bound lipoyl groups (Fig. 1). Pyruvate is rapidly decarboxylated and produces a thiamin diphosphate-bound enamine. The enamine then reacts with lipoamide to form a tetrahedral intermediate. Rearrangement results in the formation of acetyl dihydrolipoamide and thiamin diphosphate (ThDP). Due to the lack of structural data this reaction scheme is still hypothetical, but recent model studies support this
The acetyltransferase component (E2p)
In all 2-oxo acid dehydrogenase complexes, the E2 component has a multi-domain structure in which the independent folding units of the lipoyl domains, the E1/E3 binding domain and the catalytic domain are separated by flexible linkers of 20–40 amino acids (Fig. 2).
The number of lipoyl domains varies from one to three. In the complexes from Gram-negative organisms the number is usually two (H. influenza, N. meningitidis, N. gonorrhoeae, A. eutrophus) or three (E. coli, A. vinelandii). However,
The dihydrolipoamide dehydrogenase component (E3)
Crystal structures of the A. vinelandii, Pseudomonas fluorescens, P. putida and N. meningitidis enzyme are available [75, 76, 77, 78]. An excellent in-depth review on this enzyme and related members of the disulfide reductase family has been written by Williams [79]. For mechanistic aspects of the enzyme the reader is referred to this review. The enzyme consists of two identical subunits with the catalytic site located at the subunit interface. This site contains an FAD prosthetic group and a
Assembly
The PDHC from A. vinelandii is the smallest known oxo acid dehydrogenase complex. Its mass is 700 kDa, while that of the E. coli complex is 4.5 MDa. E1p and E3 are non-covalently bound to the core protein. They can be resolved from the complex by high pH and high salt treatment, respectively, and the complex can be reconstituted from the resolved components [86, 87, 88, 89]. The isolated core protein is cubic, both in the A. vinelandii and the E. coli E2p. E. coli E2p can bind 24 E1p or 24 E3
Active site coupling
One of the most interesting aspects of organization of the oxo acid dehydrogenase complexes is the role of the lipoyl domains in coupling of the many active sites. Since the work of Shepherd and Hammes [96] and Scouten et al. [97] on the measurement of distances between active sites of the complex by fluorescence energy transfer it was realized that a single lipoyl group was unable to bridge the gap of more than 4 nm between active sites as indicated by the fluorescence studies. It was then
Conclusions
Benefits of multi-enzyme catalysis are: (a) increased efficiency by limited diffusion of catalytic intermediates and the creation of microenvironments with a high substrate concentration (channeling); (b) increase in the stability of participating protein components; and (c) finer and more effective regulation.
For the pyruvate dehydrogenase complex, channeling is provided by the lipoyl domains, which increases the efficiency of each step by at least a factor of 50 [37]. Considering the
Acknowledgements
This work was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO).
References (111)
- et al.
Curr. Opin. Struct. Biol.
(1992) - et al.
Eur. J. Biochem.
(1991) - et al.
FEMS Microbiol. Lett.
(1996) - et al.
J. Biol. Chem.
(1997) - et al.
Biochem. Biophys. Res. Commun.
(1968) - et al.
J. Biol. Chem.
(1970) - et al.
FEBS Lett.
(1989) - et al.
J. Biol. Chem.
(1963) - et al.
FEBS Lett.
(1982) - et al.
J. Mol. Biol.
(1989)
J. Mol. Biol.
J. Mol. Biol.
J. Mol. Biol.
J. Mol. Biol.
Structure
J. Mol. Biol.
J. Mol. Biol.
Structure
J. Mol. Biol.
FEMS Microbiol. Lett.
J. Mol. Biol.
J. Mol. Biol.
J. Mol. Biol.
FEBS Lett.
J. Biol. Chem.
J. Mol. Biol.
Biochemistry
Biol. Chem. Hoppe-Seyler
J. Gen. Microbiol.
Eur. J. Biochem.
Eur. J. Biochem.
Eur. J. Biochem.
Eur. J. Biochem.
Eur. J. Biochem.
Eur. J. Biochem.
J. Bacteriol.
J. Med. Microbiol.
J. Bacteriol.
Mol. Microbiol.
Mol. Microbiol.
Mol. Gen. Genet.
J. Bacteriol.
Eur. J. Biochem.
Eur. J. Biochem.
Eur. J. Biochem.
Biochemistry
J. Bacteriol.
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