Mobility in pyruvate dehydrogenase complexes with multiple lipoyl domains

Machado, Rosane S.; Guest, John R.; Williamson, Michael P.

doi:10.1016/0014-5793(93)81349-5

Cited by 12 publications

(6 citation statements)

References 17 publications

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“…This is apparent from the impairment that accompanies the shortening or replacing of the residual lipoyl linker of a llip-PDH complex, and the enhancement afforded by incorporating a polyproline linker (Miles et al, 1988 ;Turner et al, 1993). High-field H1-NMR studies with variants containing from zero to nine lipoyl domains per E2p chain gave the first indication that the mobility of the lipoyl linkers relative to the domains, and hence the catalytic efficiency, is highest in the wild-type complex (Machado et al, 1993). The superiority of the 3lip-PDH complex has since been confirmed in physiological studies with stable isogenic strains that express llip-, 21ip-and 3lip-PDH complexes, in which it was concluded that lowering the lipoyl domain content adversely affects growth rate and yield (DavC et al, 1995).…”

Section: Introductionmentioning

confidence: 99%

Enzymological and physiological consequences of restructuring the lipoyl domain content of the pyruvate dehydrogenase complex of Escherichia coli

et al. 1997

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The core-forming lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli contain three tandemly repeated lipoyl domains although one lipoyl domain is apparently sufficient for full catalytic activity in vitro. Plasmids containing IPTG-inducible aceEF-lpdA operons which express multilip-PDH complexes bearing one Nterminal lipoyl domain and up to seven unlipoylated (mutant) domains per E2p chain, were constructed. Each plasmid restored the nutritional lesion of a strain lacking the PDH complex and expressed a sedimentable PDH complex, although the catalytic activities declined significantly as the number of unlipoylated domains increased above four per E2p chain. It was concluded that the extra domains protrude from the 24-meric E2p core without affecting assembly of the E l p and E3 subunits, and that the lipoyl cofactor bound to the outermost domain can participate successfully at each of the three types of active site in the assembled complex. Physiological studies with two series of isogenic strains expressing multilip-PDH complexes from modified chromosomal pdh operons (PdhR-aceEFlpdA) showed that three lipoyl domains per E2p chain is optimal and that only the outermost domain need be lipoylated for optimal activity. It is concluded that the reason for retaining three lipoyl domains is t o extend the reach of the outermost lipoyl cofactor rather than to provide extra cofactors for catalysis.

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Section: Introductionmentioning

confidence: 99%

Enzymological and physiological consequences of restructuring the lipoyl domain content of the pyruvate dehydrogenase complex of Escherichia coli

et al. 1997

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“…However, as noted in the Introduction, some roles require assembled E2 structures. Guest and colleagues have characterized a variety of assembled E. coli PDC-E2 structures for lipoyl domain roles and found the three lipoyl domains have equivalent capacities in supporting the overall PDC reaction (67)(68)(69). As in the case of S. cerevisiae PDC (27), production of mammalian E2 free of E3BP is important for distinguishing the contribution of these two lipoyl-bearing components in catalytic and regulatory processes.…”

mentioning

confidence: 99%

Assembly and Full Functionality of Recombinantly Expressed Dihydrolipoyl Acetyltransferase Component of the Human Pyruvate Dehydrogenase Complex

Yang

Song

Wagenknecht

et al. 1997

Journal of Biological Chemistry

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The dihydrolipoyl acetyltransferase (E2) component of mammalian pyruvate dehydrogenase complex (PDC) consists of 60 COOH-terminal domains as an inner assemblage and sequentially via linker regions an exterior pyruvate dehydrogenase (E1) binding domain and two lipoyl domains. Mature human E2, expressed in a protease-deficient Escherichia coli strain at 27°, was prepared in a highly purified form. Purified E2 had a high acetyltransferase activity, was well lipoylated based on its acetylation, and bound a large complement of bovine E1. Electron micrographs demonstrated that the inner core was assembled in the expected pentagonal dodecahedron shape with E1 binding around the inner core periphery.With saturating E1 and excess dihydrolipoyl dehydrogenase (E3) but no E3-binding protein (E3BP), the recombinant E2 supported the overall PDC reaction at 4% of the rate of bovine E2⅐E3BP subcomplex. The lipoates of assembled human E2 or its free bilipoyl domain region were reduced by E3 at rates proportional to the lipoyl domain concentration, but those of the E2⅐E3BP were rapidly used in a concentration-independent manner consistent with bound E3 rapidly using a set of lipoyl domains localized nearby. Given this restriction and the need for E3BP for high PDC activity, directed channeling of reducing equivalents to bound E3 must be very efficient in the complex.The recombinant E2 oligomer increased E1 kinase activity by up to 4-fold and, in a Ca 2؉ -dependent process, increased phospho-E1 phosphatase activity more than 15-fold. Thus the E2 assemblage fully provides the molecular intervention whereby a single E2-bound kinase or phosphatase molecule rapidly phosphorylate or dephosphorylate, respectively, many E2-bound E1. Thus, we prepared properly assembled, fully functional human E2 that mediated enhanced regulatory enzyme activities but, lacking E3BP, supported low PDC activity.Pyruvate dehydrogenase complexes from various sources are among the largest enzyme systems that serve strategic roles in metabolism (1, 2). The mammalian complex has a highly organized structure in which the dihydrolipoyl transacetylase (E2) 1 component has a central role in the organization and integrated chemical reactions of the complex, and supports enhanced functioning of dedicated kinase and phosphatase components (3-8). The other components of the mammalian complex required for the overall reaction are: the pyruvate dehydrogenase (E1) component, an ␣ 2 ␤ 2 tetramer present at 20 -30 copies (1); the dihydrolipoyl dehydrogenase (E3), a homodimer present in about 6 copies (9); and the E3-binding protein (E3BP) estimated at 6 -12 copies (10, 11). Dedicated and highly regulated kinase and phosphatase components control the conversion of E1 between an active (nonphosphorylated) form, E1a, and an inactive (phosphorylated) form, E1b.Mammalian PDC-E2 subunits have four flexibly connected domains and form the core of the complex in which 60 of its COOH-terminal inner (I) domains assemble into a dodecahedron-shaped structure with its other three domains ...

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“…High-field NMR studies were carried out with variants containing zero to nine lipoyl domains per E2p subunit. These studies suggest an explanation for the presence of three lipoyl domains per E2p subunit in the wild-type PDH complex that is based on the greater inherent mobility and thus potentially more efficient active-site coupling of this arrangement (170). The superiority of the three lipoyl domain-PDH complex has since been confirmed by physiological studies from which it was concluded that decreased lipoyl domain contents adversely affect growth rate and growth yield (171).…”

Section: Structures Of Lipoylated and Biotinylated Proteinsmentioning

confidence: 87%

Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation

Cronan

2008

EcoSal Plus

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SummaryTwo vitamins, biotin and lipoic acid, are essential in all three domains of life. Both coenzymes function only when covalently attached to key metabolic enzymes. There they act as "swinging arms" that shuttle intermediates between two active sites (= covalent substrate channeling) of key metabolic enzymes. Although biotin was discovered over 100 years ago and lipoic acid 60 years ago, it was not known how either coenzyme is made until recently. In Escherichia coli the synthetic pathways for both coenzymes have now been worked out for the first time.The late steps of biotin synthesis, those involved in assembling the fused rings, were welldescribed biochemically years ago, although recent progress has been made on the BioB reaction, the last step of the pathway in which the biotin sulfur moiety is inserted. In contrast, the early steps of biotin synthesis, assembly of the fatty acid-like "arm" of biotin were unknown. It has now been demonstrated that the arm is made by using disguised substrates to gain entry into the fatty acid synthesis pathway followed by removal of the disguise when the proper chain length is attained. The BioC methyltransferase is responsible for introducing the disguise and the BioH esterase for its removal.In contrast to biotin, which is attached to its cognate proteins as a finished molecule, lipoic acid is assembled on its cognate proteins. An octanoyl moiety is transferred from the octanoyl-ACP of fatty acid synthesis to a specific lysine residue of a cognate protein by the LipB octanoyl transferase followed by sulfur insertion at carbons C6 and C8 by the LipA lipoyl synthetase. Assembly on the cognate proteins regulates the amount of lipoic acid synthesized and thus there is no transcriptional control of the synthetic genes. In contrast transcriptional control of the biotin synthetic genes is wielded by a remarkably sophisticated, yet simple, system, exerted through BirA a dual function protein that both represses biotin operon transcription and ligates biotin to its cognate protein.

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Mobility in pyruvate dehydrogenase complexes with multiple lipoyl domains

Cited by 12 publications

References 17 publications

Enzymological and physiological consequences of restructuring the lipoyl domain content of the pyruvate dehydrogenase complex of Escherichia coli

Enzymological and physiological consequences of restructuring the lipoyl domain content of the pyruvate dehydrogenase complex of Escherichia coli

Assembly and Full Functionality of Recombinantly Expressed Dihydrolipoyl Acetyltransferase Component of the Human Pyruvate Dehydrogenase Complex

Biotin and Lipoic Acid: Synthesis, Attachment, and Regulation

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