The FNR-like domain of the <i>Escherichia coli</i> sulfite reductase flavoprotein component: crystallization and preliminary X-ray analysis

Gruez, Arnaud; Zeghouf, Mahel; Bertrand, J. A.; Eschenbrenner, Michel; Covès, Jacques; Fontecave, Marc; Pignol, David; Fontecilla‐Camps, J.C.

doi:10.1107/s090744499701069x

Cited by 6 publications

(3 citation statements)

References 16 publications

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“…Re®nement of both RNase Sa3 structures is under way. The high solvent content in crystal form I is not typical for protein crystals; however, similar values have been frequently observed, for example, crystals of oxygenevolving photosystem II with a solvent content of 66% (Shen & Kamiya, 2000), rotavirus protein VP6 with a solvent content of 70% (Petitpas et al, 1998), the complex of tRNAAsp with aspartyl-tRNA synthetase with a solvent content of 75% (Briand et al, 1998), FNR-like domain of the sul®te reductase¯avoprotein subunit with a solvent content of 75% (Gruez et al, 1998), glutathione S-transferase with a solvent content of 75% (Rossjohn et al, 1997) and glucosamine-6-phosphate deaminase with a solvent content of 78% (Horjales et al, 1999).…”

Section: Resultsmentioning

confidence: 55%

Purification, crystallization and preliminary X-ray analysis of two crystal forms of ribonuclease Sa3

Hlinková

Urbanikova

Krajcikova

et al. 2001

Acta Crystallogr D Biol Cryst

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RNase Sa3 produced by Streptomyces aureofaciens strain CCM 3239 belongs to the T1 family of microbial ribonucleases. It is closely related both to RNase Sa, studied in detail earlier, and to RNase Sa2 produced by the same microorganism. The most important property of RNase Sa3 is the relatively high cytotoxic activity, which was not observed for RNase Sa and Sa2. Recombinant RNase Sa3 was overexpressed in Escherichia coli and puri®ed to high homogeneity. The hanging-drop vapour-diffusion method was used for crystallization. The two crystal forms are trigonal P3 1 21 and tetragonal P4 1 2 1 2, with unit-cell parameters a = b = 64.7, c = 69.6 A Ê , = 120 and a = b = 34.0, c = 147.2 A Ê , respectively. They diffract to 2.0 and to 1.7 A Ê resolution, respectively, using synchrotron radiation. The asymmetric units of crystal forms I and II contain one molecule of the enzyme, which corresponds to V M = 3.8 A Ê 3 Da À1 with a solvent content of 68% and V M = 1.9 A Ê 3 Da À1 with a solvent content of 37%, respectively.

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Section: Resultsmentioning

confidence: 55%

Purification, crystallization and preliminary X-ray analysis of two crystal forms of ribonuclease Sa3

Hlinková

Urbanikova

Krajcikova

et al. 2001

Acta Crystallogr D Biol Cryst

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“…Previous work has demonstrated that cysK , which encodes an enzyme in L-cysteine biosynthesis, is subject to FNR regulation and identified an FNR-like domain in cysJ, which encodes a component of the sulfite reductase [16, 61]. Here, we found that loss of FNR affects the entire L-cysteine biosynthetic pathway including genes involved in the uptake and transport of sulfate ( i.e.…”

Section: Resultsmentioning

confidence: 61%

RNA-seq analysis of the influence of anaerobiosis and FNR on Shigella flexneri

et al. 2014

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BackgroundShigella flexneri is an important human pathogen that has to adapt to the anaerobic environment in the gastrointestinal tract to cause dysentery. To define the influence of anaerobiosis on the virulence of Shigella, we performed deep RNA sequencing to identify transcriptomic differences that are induced by anaerobiosis and modulated by the anaerobic Fumarate and Nitrate Reduction regulator, FNR.ResultsWe found that 528 chromosomal genes were differentially expressed in response to anaerobic conditions; of these, 228 genes were also influenced by FNR. Genes that were up-regulated in anaerobic conditions are involved in carbon transport and metabolism (e.g. ptsG, manX, murQ, cysP, cra), DNA topology and regulation (e.g. ygiP, stpA, hns), host interactions (e.g. yciD, nmpC, slyB, gapA, shf, msbB) and survival within the gastrointestinal tract (e.g. shiA, ospI, adiY, cysP). Interestingly, there was a marked effect of available oxygen on genes involved in Type III secretion system (T3SS), which is required for host cell invasion and pathogenesis. These genes, located on the large Shigella virulence plasmid, were down regulated in anaerobiosis in an FNR-dependent manner. We also confirmed anaerobic induction of csrB and csrC small RNAs in an FNR-independent manner.ConclusionsAnaerobiosis promotes survival and adaption strategies of Shigella, while modulating virulence plasmid genes involved in T3SS-mediated host cell invasion. The influence of FNR on this process is more extensive than previously appreciated, although aside from the virulence plasmid, this transcriptional regulator does not govern expression of genes on other horizontally acquired sequences on the chromosome such as pathogenicity islands.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-438) contains supplementary material, which is available to authorized users.

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“…The crystal structure of SiR‐HP has been solved at 1.6 Å resolution [10] but structural information about SiR‐FP is not available yet despite preliminary results obtained with the isolated FAD‐binding domain [11]. The FMN and FAD cofactors are involved in different diaphorase catalytic activities used for the in‐vitro characterization of the protein [1,3,12,13].…”

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confidence: 99%

³¹P Nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase

Evrard

Zeghouf

Fontecave

et al. 1999

European Journal of Biochemistry

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SiR-FP60, the monomeric form of the Escherichia coli sulfite reductase flavoprotein component (SiR-FP), has been analysed by 31 P-NMR spectroscopy. This protein was reported previously as a reliable simplified model for native SiR-FP [Zeghouf, M., Fontecave, M., Macherel, D., & Cove Ás, J. (1998) Biochemistry 37, 6117±6123]. SiR-FP60 was examined in its native form, as a complex with NADP + and after monoelectronic reduction either with NADPH or dithionite. In these latter cases, the stabilized FMN semiquinone radical offers a natural and internal paramagnetic probe. The paramagnetic effect of added manganese was also studied. In each case, the NMR parameters were extracted from digitalized data by a deconvolution procedure and compared with those obtained previously with cytochrome P450 reductase. Evolution of the NMR parameters and of calculated relaxation rate constants upon biochemical modifications of SiR-FP60 led us to propose that the reactive center is more compact than the one of cytochrome P450 reductase, with the redox components, FMN, FAD and NADPH, in a tighter spatial arrangement, close to the protein surface. This underlies some subtle differences between the two proteins for which a very similar overall structure is likely considering their common genetic origin and common operating cycle.Keywords: sulfite reductase; cytochrome P450 reductase; 31 P-NMR; protein structure; Escherichia coli.Escherichia coli sulfite reductase (SiR) is a multimeric and soluble hemoflavoprotein with a molecular mass of 780 kDa and an a 8 b 4 quaternary structure. Subunit a is the flavoprotein component (SiR-FP) of the enzyme. It contains one NADPHbinding domain and two distinct flavinic domains: an N-terminal flavodoxin-like domain that binds one FMN and a C-terminal ferredoxin-NADP + reductase-like, FAD-containing domain [1,2]. This was strongly suggested from sequence comparisons, limited proteolysis experiments and expression of the isolated FMNbinding domain [1±3]. During the physiological turnover leading to reduction of sulfite, the first event is an hydride transfer from NADPH to bound FAD. Electrons are then transferred to FMN before being transferred one at a time to the b-subunits, which form the hemoprotein component (SiR-HP), where the six-electron reduction of sulfite to sulfide takes place [4,5].These characteristics, i.e. a bi-flavin domain, NADPH as the electron donor and, a metal center as the electron acceptor, are shared by all the members of the SiR-FP family that includes mammalian NADPH-cytochrome P450 reductases, nitric oxide synthase (NOS) and Bacillus megaterium cytochrome P450 (P450BM-3) [6,7]. Among these proteins, only the structure of cytochrome P450 reductase is known [8] but amino-acid sequence analysis and structural alignment with flavodoxin and ferredoxin reductase predict an overall structure very similar to that of cytochrome P450 reductase for all the members of the family [8]. The hypothesis that SiR-FP belongs to this family of proteins was strengthened by the recent demonstrati...

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The FNR-like domain of the Escherichia coli sulfite reductase flavoprotein component: crystallization and preliminary X-ray analysis

Cited by 6 publications

References 16 publications

Purification, crystallization and preliminary X-ray analysis of two crystal forms of ribonuclease Sa3

Purification, crystallization and preliminary X-ray analysis of two crystal forms of ribonuclease Sa3

RNA-seq analysis of the influence of anaerobiosis and FNR on Shigella flexneri

³¹P Nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase

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The FNR-like domain of the Escherichia coli sulfite reductase flavoprotein component: crystallization and preliminary X-ray analysis

Cited by 6 publications

References 16 publications

Purification, crystallization and preliminary X-ray analysis of two crystal forms of ribonuclease Sa3

Purification, crystallization and preliminary X-ray analysis of two crystal forms of ribonuclease Sa3

RNA-seq analysis of the influence of anaerobiosis and FNR on Shigella flexneri

31P Nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase

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³¹P Nuclear magnetic resonance study of the flavoprotein component of the Escherichia coli sulfite reductase