Polypeptide chain stoicheiometry in the self‐assembly of the pyruvate dehydrogenase multienzyme complex of <i>Escherichia coli</i>

Perham, Richard N.; Hooper, Elizabeth A.

doi:10.1016/0014-5793(77)80965-4

Cited by 15 publications

(7 citation statements)

References 13 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Another problem presents the optimum number of El dimers which can be bound to the complex from E. coli. Direct determinations of chain stoichiometry of isolated complexes and reconstitution experiments of El with E2E3 subcomplexes performed by the group of Perham [8,33,49] are very close to the values reported here for the A . cinelandii complex, when they are corrected for the molecular mass of 66 kDa of E, .…”

Section: Discussionsupporting

confidence: 86%

The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii

Bosma¹,

Kok

Westphal

et al. 1984

European Journal of Biochemistry

View full text Add to dashboard Cite

An improved purification procedure of the pyruvate dehydrogenase complex of Azotobucter vinelundii is described. This procedure minimizes losses of components and results in the isolation of the pure complex with a specific activity of 15 -19 U/mg and an overall yield of 40 %.The chain ratio of the three components was determined by covalent modification of the lysine residues with trinitrobenzene sulfonic acid, followed by separation of the components on sodium dodecyl sulfate gels. These determinations yielded an average chain ratio of 1.3:3 :0.5 for El :E, :E, respectively. Based on Ez this corresponds with a minimum molecular mass of approximately 216 kDa. Because the molecular mass of the complex has been determined previously to be 800 50 kDa, it is concluded that the complex as isolated from A . vinelundii is based on a tetramer of E2 chains.The complex can be resolved into its individual components, which can be recombined to yield a fully active complex. Titration of E,E, subcomplexes with El resulted in maximum complex activity at an EJE, ratio of 1.5 -1.6. Similar titrations of EIE, subcomplexes with E, resulted in maximum activity at an E,/E, ratio of 0.45 -0.55. From these experiments it is concluded that the complex has maximum activity with a composition of three El dimers, one E, tetramer and one E, dimer. With excess of either El or E, a decrease in activity is observed which indicates competition between these components for binding sites on E,.Asshownbefore a core of 24 E, chains, arranged in a cube with 432 symmetry [l]. Estimates of the molecular mass of this complex vary within 3 -6 MDa [2,4 -71.There is a long-lasting discussion on the stoichiometry of the E. coli complex; chain ratios of 2:2:1 [6], 2:l:l [8] and I :I :I [9] have been proposed. It has been recognized that the amounts of El and E, are not constant in the organism, that preparations of the complex show heterogeneity, and that the chain ratio may change during purification due to losses of these components [5,9 -131. Nevertheless, the E2 core has an inborn capacity for the binding of El and E, dimers. The discrepancy between the reports of several investigators is therefore probably due to a different interpretation of results as obtained by various methods.The pyruvate dehydrogenase complex as isolated from the gram-negative bacterium Azotobucter vinelandii is much smaller than the E. coli complex [14]. Its sedimentation coefficient is 17 -19 S, that of E. coli PDC is 53 -60 S [2,4,7,10]. However, a 17 -20-S form of the E. coli PDC has been observed [7,10] and we have shown that the A . vinelandii complex can be converted into a 56-S form, resembling the E. coli PDC on electron micrographs [15]. 542We have shown recently [I61 that the 56-S form of the A . Ginelandii PDC is an octamer of a 750 -850-kDa particle. The tendency to aggregate is especially pronounced in the isolated E, component; its appearance on electron micrographs is similar to that of E, from E. coli. By sedimentation and light scattering studies it wa5 shown...

show abstract

Section: Discussionsupporting

confidence: 86%

The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii

Bosma¹,

Kok

Westphal

et al. 1984

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Thiamine pyrophosphate is bound to E1 and participates in pyruvate decarboxylation; lipoate is covalently attached to a conserved lysine residue of E2 and mediates translocation of intermediates between the active sites of E1, E2, and E3, while FAD and NAD + are required for reoxidation of dihydrolipoate by E3. A complex architecture, involving up to 60 copies per subunit in one PDH complex, enables efficient coordination of the E1, E2, and E3 activities and is, in some organisms, the result of self-assembly ( 2 , 3 ). In other organisms, assembly requires additional proteins ( 4 ).…”

Section: Introductionmentioning

confidence: 99%

Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of Saccharomyces cerevisiae

Kozak

Rossum

Luttik

et al. 2014

mBio

View full text Add to dashboard Cite

The energetic (ATP) cost of biochemical pathways critically determines the maximum yield of metabolites of vital or commercial relevance. Cytosolic acetyl coenzyme A (acetyl-CoA) is a key precursor for biosynthesis in eukaryotes and for many industrially relevant product pathways that have been introduced into Saccharomyces cerevisiae, such as isoprenoids or lipids. In this yeast, synthesis of cytosolic acetyl-CoA via acetyl-CoA synthetase (ACS) involves hydrolysis of ATP to AMP and pyrophosphate. Here, we demonstrate that expression and assembly in the yeast cytosol of an ATP-independent pyruvate dehydrogenase complex (PDH) from Enterococcus faecalis can fully replace the ACS-dependent pathway for cytosolic acetyl-CoA synthesis. In vivo activity of E. faecalis PDH required simultaneous expression of E. faecalis genes encoding its E1α, E1β, E2, and E3 subunits, as well as genes involved in lipoylation of E2, and addition of lipoate to growth media. A strain lacking ACS that expressed these E. faecalis genes grew at near-wild-type rates on glucose synthetic medium supplemented with lipoate, under aerobic and anaerobic conditions. A physiological comparison of the engineered strain and an isogenic Acs+ reference strain showed small differences in biomass yields and metabolic fluxes. Cellular fractionation and gel filtration studies revealed that the E. faecalis PDH subunits were assembled in the yeast cytosol, with a subunit ratio and enzyme activity similar to values reported for PDH purified from E. faecalis. This study indicates that cytosolic expression and assembly of PDH in eukaryotic industrial microorganisms is a promising option for minimizing the energy costs of precursor supply in acetyl-CoA-dependent product pathways.

show abstract

“…He was one of the first to use the approach, now termed 'integrative structural biology', in which a wide variety of techniques are combined to determine a molecular structure. (43,47,53) and peripheral subunit binding-domains (40,44,60) of different 2-oxo acid dehydrogenase complexes; and the X-ray crystal structure of the E2 catalytic core (50). He then integrated the results to solve the structures of different protein sub-complexes and those of interacting domains (46,59).…”

Section: Back To Science -Discoveries In Mechanistic Enzymologymentioning

confidence: 99%

Richard Nelson Perham. 27 April 1937—14 February 2015

Berry

Radford

2018

Biogr. Mems Fell. R. Soc.

View full text Add to dashboard Cite

Richard Nelson Perham, FRS, FMedSci, FRSA, was a British Professor of Structural Biochemistry. He undertook his academic career at the University of Cambridge, holding positions as Lecturer, Reader, Chair and Head of the Department of Biochemistry, as well as Master of St John's College. Perham published close to 300 scientific papers on protein structure and function, with a focus on mechanistic enzymology, particularly how large multienzyme complexes and flavin-containing enzymes work. He is most renowned for determining how reactive intermediates are transferred between enzyme active sites, for alterations of coenzyme and substrate specificity by his pioneering use of protein engineering and for developing protein display methodologies. Married to Nancy Lane-Perham and with their two children, Perham enjoyed a full and active life in Cambridge and St John's College. He was a keen participator and supporter of sport and enjoyed art, literature, theatre and music. Perham was a vocal and active champion of equal opportunity in education. His legacy to science is a greater understanding of how enzymes work. His legacy to scientists is as a role model of how to attain the highest levels of achievement whilst maintaining a sense of personal modesty and keen support for others.

show abstract

Polypeptide chain stoicheiometry in the self‐assembly of the pyruvate dehydrogenase multienzyme complex of Escherichia coli

Cited by 15 publications

References 13 publications

The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii

The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii

Engineering Acetyl Coenzyme A Supply: Functional Expression of a Bacterial Pyruvate Dehydrogenase Complex in the Cytosol of Saccharomyces cerevisiae

Richard Nelson Perham. 27 April 1937—14 February 2015

Contact Info

Product

Resources

About