Crystal structure of YnjE from Escherichia coli,a sulfurtransferase with three rhodanese domains

Rhodaneses/sulfurtransferases are ubiquitous enzymes that catalyze the transfer of sulfane sulfur from a donor molecule to a thiophilic acceptor via an active site cysteine that is modified to a persulfide during the reaction. Here, we present the first crystal structure of a triple‐domain rhodanese‐like protein, namely YnjE from Escherichia coli, in two states where its active site cysteine is either unmodified or present as a persulfide. Compared to well‐characterized tandem domain rhodaneses, which are composed of one inactive and one active domain, YnjE contains an extra N‐terminal inactive rhodanese‐like domain. Phylogenetic analysis reveals that YnjE triple‐domain homologs can be found in a variety of other γ‐proteobacteria, in addition, some single‐, tandem‐, four and even six‐domain variants exist. All YnjE rhodaneses are characterized by a highly conserved active site loop (CGTGWR) and evolved independently from other rhodaneses, thus forming their own subfamily. On the basis of structural comparisons with other rhodaneses and kinetic studies, YnjE, which is more similar to thiosulfate:cyanide sulfurtransferases than to 3‐mercaptopyruvate:cyanide sulfurtransferases, has a different substrate specificity that depends not only on the composition of the active site loop with the catalytic cysteine at the first position but also on the surrounding residues. In vitro YnjE can be efficiently persulfurated by the cysteine desulfurase IscS. The catalytic site is located within an elongated cleft, formed by the central and C‐terminal domain and is lined by bulky hydrophobic residues with the catalytic active cysteine largely shielded from the solvent.     

Escherichia coli GlpE is a prototype sulfurtransferase for the single-domain rhodanese homology superfamily.

BACKGROUND: Rhodanese domains are structural modules occurring in the three major evolutionary phyla. They are found as single-domain proteins, as tandemly repeated modules in which the C-terminal domain only bears the properly structured active site, or as members of multidomain proteins. Although in vitro assays show sulfurtransferase or phosphatase activity associated with rhodanese or rhodanese-like domains, specific biological roles for most members of this homology superfamily have not been established. RESULTS: Eight ORFs coding for proteins consisting of (or containing) a rhodanese domain bearing the potentially catalytic Cys have been identified in the Escherichia coli K-12 genome. One of these codes for the 12-kDa protein GlpE, a member of the sn-glycerol 3-phosphate (glp) regulon. The crystal structure of GlpE, reported here at 1.06 A resolution, displays alpha/beta topology based on five beta strands and five alpha helices. The GlpE catalytic Cys residue is persulfurated and enclosed in a structurally conserved 5-residue loop in a region of positive electrostatic field. CONCLUSIONS: Relative to the two-domain rhodanese enzymes of known three-dimensional structure, GlpE displays substantial shortening of loops connecting alpha helices and beta sheets, resulting in radical conformational changes surrounding the active site. As a consequence, GlpE is structurally more similar to Cdc25 phosphatases than to bovine or Azotobacter vinelandii rhodaneses. Sequence searches through completed genomes indicate that GlpE can be considered to be the prototype structure for the ubiquitous single-domain rhodanese module.     

Solution structures and backbone dynamics of Escherichia coli rhodanese PspE in its sulfur-free and persulfide-intermediate forms: implications for the catalytic mechanism of rhodanese

Rhodanese catalyzes the sulfur-transfer reaction that transfers sulfur from thiosulfate to cyanide by a double-displacement mechanism, in which an active cysteine residue plays a central role. Previous studies indicated that the phage-shock protein E (PspE) from Escherichia coli is a rhodanese composed of a single active domain and is the only accessible rhodanese among the three single-domain rhodaneses in E. coli. To understand the catalytic mechanism of rhodanese at the molecular level, we determined the solution structures of the sulfur-free and persulfide-intermediate forms of PspE by nuclear magnetic resonance (NMR) spectroscopy and identified the active site by NMR titration experiments. To obtain further insights into the catalytic mechanism, we studied backbone dynamics by NMR relaxation experiments. Our results demonstrated that the overall structures in both sulfur-free and persulfide-intermediate forms are highly similar, suggesting that no significant conformational changes occurred during the catalytic reaction. However, the backbone dynamics revealed that the motional properties of PspE in its sulfur-free form are different from the persulfide-intermediate state. The conformational exchanges are largely enhanced in the persulfide-intermediate form of PspE, especially around the active site. The present structural and biochemical studies in combination with backbone dynamics provide further insights in understanding the catalytic mechanism of rhodanese.     

Properties of thermostable HtpR-mutant of Escherichia coli

A thermoresistant htpR mutant having a decreased level of proteolytic activity has been selected in E. coli strain K802 after the directed mutagenesis in vivo. The mutation results in the bacteriophage T7 RNA-polymerase stability, aminoglycosidephosphotransferase stability as well as in the decrease in the rate of proteolytic degradation of cytoplasmic proteins during the heat shock. The obtained mutant strain can, probably be used as a host for alien polypeptides production.     

Properties of gamma-aminobutyraldehyde dehydrogenase from Escherichia coli

gamma-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source. The enzyme has an Mr of 195,000 +/- 10,000 in its dimeric form with an Mr of 95,000 +/- 1,000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity. Km values are 31.3 +/- 6.8 microM and 53.8 +/- 7.4 microM for delta-1-pyrroline and NAD+, respectively. An inhibitory capacity for NADH is also shown using the purified enzyme.     

1H, 13C and 15N resonance assignments of rhodanese GlpE from Escherichia coli

Rhodanese catalyzes the sulfur-transfer reaction in which a sulfur atom is transferred from thiosulfate to cyanide by a double-displacement mechanism. During the reaction, a persulfide-intermediate form of rhodanese is generated by the reaction of a conserved active cysteine residue with thiosulfate. Escherichia coli GlpE is a prototype for the single-domain rhodanese superfamily. Though there are some studies on rhodaneses, the molecular mechanism of the catalytic activity of rhodaneses is still unclear. Herein, we report the resonance assignments of (1)H, (13)C and (15)N atoms of E. coli GlpE, which provides the basis for further structural, dynamic and functional studies of rhodaneses using NMR technique.     

Overexpression of the rhodanese PspE, a single cysteine-containing protein, restores disulphide bond formation to an Escherichia coli strain lacking DsbA

Escherichia coli uses the DsbA/DsbB system for introducing disulphide bonds into proteins in the cell envelope. Deleting either dsbA or dsbB or both reduces disulphide bond formation but does not entirely eliminate it. Whether such background disulphide bond forming activity is enzyme-catalysed is not known. To identify possible cellular factors that might contribute to the background activity, we studied the effects of overexpressing endogenous proteins on disulphide bond formation in the periplasm. We find that overexpressing PspE, a periplasmic rhodanese, partially restores substantial disulphide bond formation to a dsbA strain. This activity depends on DsbC, the bacterial disulphide bond isomerase, but not on DsbB. We show that overexpressed PspE is oxidized to the sulphenic acid form and reacts with substrate proteins to form mixed disulphide adducts. DsbC either prevents the formation of these mixed disulphides or resolves these adducts subsequently. In the process, DsbC itself gets oxidized and proceeds to catalyse disulphide bond formation. Although this PspE/DsbC system is not responsible for the background disulphide bond forming activity, we suggest that it might be utilized in other organisms lacking the DsbA/DsbB system.     

Characterization of a 12-kilodalton rhodanese encoded by glpE of Escherichia coli and its interaction with thioredoxin

Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese from Escherichia coli. Well-characterized rhodaneses are comprised of two structurally similar ca. 15-kDa domains. Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses. The glpE gene, a member of the sn-glycerol 3-phosphate (glp) regulon of E. coli, encodes the 12-kDa rhodanese. As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. The K(m)s for SSO(3)(2-) and CN(-) were 78 and 17 mM, respectively. The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer. GlpE exhibited a k(cat) of 230 s(-1). Thioredoxin 1 from E. coli, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparent K(m) of 34 microM when thiosulfate was near its K(m), suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases. The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited ( approximately 17%). This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases. GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins (http://www.ncbi.nlm.nih.gov/COG/) for which sulfurtransferase activity has been confirmed.     

Properties of an Escherichia coli mutant deficient in phosphoenolpyruvate carboxylase catalytic activity.

A mutant Escherichia coli (Ppcc-) which was unable to grow on glucose as a sole carbon source was isolated. This mutant had very low levels of phosphoenolpyruvate carboxylase activity (approximately 5% of the wild type). Goat immunoglobulin G prepared against wild-type phosphoenolypyruvate carboxylase cross-reacted with the Ppcc- enzyme. The amount of enzyme protein in the mutant cells was similar to that found in wild-type cells, but it had greatly diminished specific activity. The catalytically less active mutant enzyme retained the ability to interact with fructose 1,6-bisphosphate, but did not exhibit stabilization of the tetrameric form by aspartate. The pI of the mutant protein was lower (4.9) than that of the wild-type protein (5.1). After electrophoresis and immunoblotting of the partially purified protein, several immunostaining bands were seen in addition to the main enzyme band. A novel method for showing that these bands represented proteolytic fragments of phosphoenolpyruvate carboxylase was developed.     

Properties of purified ribonuclease P from Escherichia coli

The purified protein moiety of ribonuclease P (EC 3.1.26.5) from Escherichia coli, a single polypeptide of molecular weight approximately 17 500, has not catalytic activity by itself on several RNA substrates. However, when it is marked in vitro with an RNA species called M1 RNA, RNase P activity is reconstituted. The rate at which the purified RNase P cleaves any particular tRNA precursor molecule depends on the identity of that tRNA precursor.     

Properties of Escherichia coli mutants lacking membrane-derived oligosaccharides

Membrane-derived oligosaccharides (MDO) consist of branched substituted beta-glucan chains and are present in the periplasmic space of Escherichia coli and other gram-negative bacteria. A procedure for the isolation of mutants defective in MDO synthesis is described. Their phenotype was compared with a mdoA mutant previously identified, and they are mapped in the mdoA region. Mutants lacking MDO showed imparied chemotaxis on tryptone swarm plates, a reduced number of flagella, and an enhanced expression of the OmpC porin. Revertants able to form swarm rings again had regained the ability to synthesize MDO and showed the wild-type porin pattern. A second group of chemotactic revertants were mutated in the ompB gene region involved in osmoregulation, and they were still devoid of MDO. These findings provide evidence for a link between MDO biosynthesis and other functions of E. coli related to its adaptation to the environment.     

Properties of electroporation-mediated DNA transfer in Escherichia coli

Efficient and reproducible DNA-transfection was attained in E. coli, by electroporation. The yield of the transfectants was affected by pretreatment of the recipient cells as well as by the composition of the electroporation medium. Using a single pulse procedure, relationships among the electrical parameters, the transfection efficiency, and the cellular viability were investigated in 10 mM Tris-HCl buffer (pH 7.5) containing 5% sucrose. Certain sodium salts (e.g., citrate, phosphate, and sulfate) were promotive, whereas Mg2+, DEAE-dextran, and polyvinylpyrrolidone were inhibitory to the transfection. Heterologous nucleic acids (native DNA, denatured DNA, and tRNA) exerted only a marginal effect on transfection with a viral replicative-form DNA. The efficiency of DNA transfer was affected by culture conditions, and bacteria grown at a higher temperature were more competent. The electroporation system was more efficient than an improved CaCl2 method, not only in transfection with viral single- and double-stranded DNAs, but also in transformation with plasmid DNAs.     

1H, 13C and 15N resonance assignments of the rhodanese domain of YgaP from Escherichia coli

Rhodanese domain is a ubiquitous structural module commonly found in bacterial, archaeal and eukaryotic cells. Growing evidence indicates that rhodanese domains act as the carrier of reactive sulfur atoms by forming persulfide intermediates in distinct metabolic pathways. YgaP, a membrane protein consisting of a rhodanese domain and a C-terminal transmembrane segment, is the only membrane-associated rhodanese in Escherichia coli. Herein, we report the resonance assignments of ¹H, ¹³C and ¹⁵N atoms of rhodanese domain of YgaP. Totally, chemical shifts of more than 95% of the atoms were assigned.