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The 2.6-A refined structure of the Escherichia coli recombinant Saccharomyces cerevisiae flavocytochrome b2-sulfite complex.

ABSTRACT: Flavocytochrome b2 from Saccharomyces cerevisiae catalyzes the oxidation of L-lactate to pyruvate and the electron transfer to cytochrome c in the mitochondrial intermembrane space. It is a homotetramer with a molecular weight of 4 x 58 kDa, each monomer of which is composed of 2 distinct domains, the one carrying FMN and the other, a "b5-like" heme. The native structure has been described at a resolution of 2.4 A (Xia ZX, Mathews FS, 1990, J Mol Biol 212:837-863). The heme domains protrude from the central body of the tetramer consisting of the 4 FMN binding domains. Because only 2 heme domains are visible in the electron density map, the other 2 are probably disordered. We crystallized the Escherichia coli recombinant flavocytochrome b2 from S. cerevisiae inhibited by sulfite. Although the crystals were obtained under very different conditions from those of the pyruvate-containing native enzyme, they were found to be isostructural (P 3(2) 2 1, a = b = 164.5 A, c = 114.0 A). The 2.6-A X-ray structure was extensively refined with X-PLOR (R = 17.3%), which made it possible to describe in detail the recombinant flavocytochrome b2 molecular structure. There exist few differences between the native and recombinant structures, in line with the fact that they show similar kinetic behavior, and they further confirm the intrinsic mobility of the heme domain (Labeyrie F, Beloil JC, Thomas MA, 1988, Biochim Biophys Acta 953:134-141). This structure will be used as a starting model in the structural resolution of flavocytochrome b2 point mutants.

SUBMITTER: Tegoni M

PROVIDER: S-EPMC2142803 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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The 2.6-A refined structure of the Escherichia coli recombinant Saccharomyces cerevisiae flavocytochrome b2-sulfite complex.

Tegoni M M Cambillau C C

Protein science : a publication of the Protein Society 19940201 2

Flavocytochrome b2 from Saccharomyces cerevisiae catalyzes the oxidation of L-lactate to pyruvate and the electron transfer to cytochrome c in the mitochondrial intermembrane space. It is a homotetramer with a molecular weight of 4 x 58 kDa, each monomer of which is composed of 2 distinct domains, the one carrying FMN and the other, a "b5-like" heme. The native structure has been described at a resolution of 2.4 A (Xia ZX, Mathews FS, 1990, J Mol Biol 212:837-863). The heme domains protrude from ...[more]

PMID: 8003966

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Project description:The HIS3+ gene of Saccharomyces cerevisiae was overexpressed in Escherichia coli and the recombinant imidazoleglycerol-phosphate dehydratase (IGPD) purified to homogeneity. Laser-desorption and electrospray m.s. indicated a molecular ion within 2 units of that expected (23833.3) on the basis of the protein sequence, with about half of the polypeptide lacking the N-terminal formylmethionine residue. IGPD initially purified as an apoprotein was catalytically inactive and mainly a trimer of M(r) 70,000. Addition of Mn2+ (but not Mg2+) caused this to assemble to an active (40 units/mg) enzyme (Mn-IGPD) comprising of 24 subunits (M(r) 573,000) and containing 1.35 +/- 0.1 Mn atoms/polypeptide subunit. An enzyme with an identical activity and metal content was also obtained when the fermenter growth medium of recombinant Escherichia coli was supplemented with MnCl2, and IGPD was purified through as Mn-IGPD rather than as the apoenzyme and assembled in vitro. Inhibition by EDTA indicated that the intrinsic Mn2+ was essential for activity. The retention of activity over time after dilution to very low concentrations of enzyme (< 20 nM) indicated that the metal remained in tight association with the protein. A novel continuous assay method was developed to facilitate the kinetic characterization of Mn-IGPD. At pH 7.0, the Km for IGP was 0.10 +/- 0.02 mM and the Ki value for inhibition by 1,2,4-triazole, 0.12 +/- 0.02 mM. In contrast with other reports, thiols had no influence on catalytic activity. The activity of Mn-IGPD varied with enzyme concentration in such a way as to suggest that it dissociates to a less active form at very low concentrations. Significant inhibition by the product, imidazole acetol phosphate, was inferred from the shape of the progress curve. Titration with, the potent competitive inhibitor, 2-hydroxy-3-(1,2,4-triazol-1-yl)propyl phosphonate indicated that Mn-IGPD contained 0.9 +/- 0.1 catalytic sites/protomer. The activity nearly doubled in the presence of high concentrations of Mn2+; the apparent Ks for stimulation was 20 microM. The basis of this effect was obscure, since there was no corresponding increase in the titre of active sites. Neither was there a discernable shift in the values of Km or Ki (above), although exogenous Mn2+ did reduce the optimum pH for kcat, from 7.2 to 6.8. On the basis of a single site/subunit, the maximum rate of catalytic turnover at 30 degrees C was 32 s-1.

Project description:BACKGROUND: Heterologous microbial production of rare plant terpenoids of medicinal or industrial interest is attracting more and more attention but terpenoid yields are still low. Escherichia coli and Saccharomyces cerevisiae are the most widely used heterologous hosts; a direct comparison of both hosts based on experimental data is difficult though. Hence, the terpenoid pathways of E. coli (via 1-deoxy-D-xylulose 5-phosphate, DXP) and S. cerevisiae (via mevalonate, MVA), the impact of the respective hosts metabolism as well as the impact of different carbon sources were compared in silico by means of elementary mode analysis. The focus was set on the yield of isopentenyl diphosphate (IPP), the general terpenoid precursor, to identify new metabolic engineering strategies for an enhanced terpenoid yield. RESULTS: Starting from the respective precursor metabolites of the terpenoid pathways (pyruvate and glyceraldehyde-3-phosphate for the DXP pathway and acetyl-CoA for the MVA pathway) and considering only carbon stoichiometry, the two terpenoid pathways are identical with respect to carbon yield. However, with glucose as substrate, the MVA pathway has a lower potential to supply terpenoids in high yields than the DXP pathway if the formation of the required precursors is taken into account, due to the carbon loss in the formation of acetyl-CoA. This maximum yield is further reduced in both hosts when the required energy and reduction equivalents are considered. Moreover, the choice of carbon source (glucose, xylose, ethanol or glycerol) has an effect on terpenoid yield with non-fermentable carbon sources being more promising. Both hosts have deficiencies in energy and redox equivalents for high yield terpenoid production leading to new overexpression strategies (heterologous enzymes/pathways) for an enhanced terpenoid yield. Finally, several knockout strategies are identified using constrained minimal cut sets enforcing a coupling of growth to a terpenoid yield which is higher than any yield published in scientific literature so far. CONCLUSIONS: This study provides for the first time a comprehensive and detailed in silico comparison of the most prominent heterologous hosts E. coli and S. cerevisiae as terpenoid factories giving an overview on several promising metabolic engineering strategies paving the way for an enhanced terpenoid yield.

Dataset Information

The 2.6-A refined structure of the Escherichia coli recombinant Saccharomyces cerevisiae flavocytochrome b2-sulfite complex.

Publications

The 2.6-A refined structure of the Escherichia coli recombinant Saccharomyces cerevisiae flavocytochrome b2-sulfite complex.

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