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An MsbA deletion mutant DeltaC21 that lacks the two C-terminal alpha-helices was expressed in Escherichia coli strain C41 and purified by metal-affinity and gel-filtration chromatography. Purified DeltaC21 retained 26% of the activity of the wild-type ATPase and had a similar binding affinity to fluorescent nucleotide derivatives. Although crystals of wild-type MsbA complexed with adenosine 5'-(beta,gamma-imido)triphosphate could not be obtained, crystals of DeltaC21 that diffracted to 4.5 A resolution were obtained. The preliminary DeltaC21 structure had the outward-facing conformation, in contrast to the previously reported E. coli MsbA structure. This result suggests that deletion of the C-terminal alpha-helices may play a role in facilitating the outward-facing nucleotide-bound crystal structure of EcMsbA.

A major factor in removing RNA primers during the processing of Okazaki fragments is DNA polymerase I (Pol I). Pol I is thought to remove the RNA primers and to fill the resulting gaps simultaneously. RNase H, encoded by rnh genes, is another factor in removing the RNA primers, and there is disagreement with respect to the essentiality of both the polA and rnh genes. In a previous study, we looked for the synthetic lethality of paralogs in Bacillus subtilis and detected several essential doublet paralogs, including the polA ypcP pair. YpcP consists of only the 5'-3' exonuclease domain. In the current study, we first confirmed that the polA genes of both Escherichia coli and B. subtilis could be completely deleted. We found that the 5'-3' exonuclease activity encoded by either polA or ypcP xni was required for the growth of B. subtilis and E. coli. Also, the 5'-3' exonuclease activity of Pol I was indispensable in the cyanobacterium Synechococcus elongatus. These results suggest that a 5'-3' exonuclease activity is essential in these organisms. Our success in constructing a B. subtilis strain that lacked all RNase H genes indicates that the enzymatic activity is dispensable, at least in the wild type. Increasing the 5'-3' exonuclease activity partially compensated for a defective phenotype of an RNase H-deficient mutant, suggesting cooperative functions for the two enzyme systems. Our search for the distribution of the 5'-3' exonuclease domain among 250 bacterial genomes resulted in the finding that all eubacteria, but not archaea, possess this domain.

Endopolygalacturonases (EC 3.2.1.15) catalyze random hydrolysis of the alpha-1,4 glycosidic linkages in polygalacturonic acid, a component of pectin. Previously, we reported crystal structures of endogenously produced Stereum purprureum endopolygalacturonase I (endoPG I), both in its native form and complexed with its product, galacturonate. However, the substrate-binding mechanism of endoPG I is still unclear, because crystals have not yet been obtained with a substrate analog, or with mutant enzymes that can bind substrates. We describe here an expression system using Escherichia coli and a purification method to prepare functionally active endoPG I for such mutation and crystallographic studies. Expression in E. coli strain Origami (DE3) provided a soluble and active enzyme with proper disulfide bond formation, whereas the enzyme expressed in BL21 (DE3) was localized in inclusion bodies. A sufficient amount of recombinant endoPG I produced by Origami (DE3) was purified by a single-step procedure using cation exchange chromatography. The specific activity of recombinant endoPG I was equivalent to that of the enzyme produced by S. purpureum. Recombinant endoPG I was crystallized under the same conditions as those used for the native enzyme produced by S. purpureum. The crystals diffracted beyond 1.0 A resolution with synchrotron radiation.

Escherichia coli B gamma-glutamylcysteine synthetase (gammaGCS) catalyzes the ATP-dependent coupling of L-Glu and L-Cys to form the glutathione precursor gamma-L-Glu-Cys and is a target for development of potential therapeutic agents. By introducing four point mutations of surface-exposed cysteine residues to serine, the gammaGCS was purified to homogeneity; single crystals have been obtained using the hanging-drop vapour-diffusion method with sodium formate. The gammaGCS crystal diffracted to 2.8 A and belongs to space group R3, with unit-cell parameters a = b = 326.7, c = 103.9 A.

Ribosome recycling factor (RRF) plays a central role during the recycling of ribosomes in the final step of protein biosynthesis in prokaryotes and is therefore a favourable target for the development of new antibiotics. The crystal structure of Escherichia coli RRF has been reported to have an open L-shaped conformation, while other RRFs from thermophilic bacteria have a strict L-shaped conformation [Yun et al. (2000), Acta Cryst. D56, 84-85]. Wild-type E. coli RRF has so far not been crystallized free from bound detergent. Here, a mutant of RRF, Arg132Gly, has been crystallized without any detergent. A complete data set from a crystal of this mutant obtained by the hanging-drop vapour-diffusion method has been collected at 2.2 A resolution using synchrotron radiation at 100 K. The crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 46.02, b = 49.27, c = 49.37 A, beta = 110.1 degrees. The currently refined structure indicates that RRF has a tRNA-like L-shaped conformation.

An MsbA deletion mutant ΔC21 that lacks the two C-terminal α-helices was expressed in Escherichia coli strain C41 and purified by metal-affinity and gel-filtration chromatography. Purified ΔC21 retained 26% of the activity of the wild-type ATPase and had a similar binding affinity to fluorescent nucleotide derivatives. Although crystals of wild-type MsbA complexed with adenosine 5'- (β,γ-imido)triphosphate could not be obtained, crystals of ΔC21 that diffracted to 4.5 A resolution were obtained. The preliminary ΔC21 structure had the outward-facing conformation, in contrast to the previously reported E. coli MsbA structure. This result suggests that deletion of the C-terminal α-helices may play a role in facilitating the outward-facing nucleotide-bound crystal structure of EcMsbA. [ABSTRACT FROM AUTHOR]

γ-Glutamylcysteine synthetase (γGCS), a rate-limiting enzyme in glutathione biosynthesis, plays a central role in glutathione homeostasis and is a target for development of potential therapeutic agents against parasites and cancer. We have determined the crystal structures of Escherkhia coli γGCS unliganded and complexed with a sulfoximine-based transition-state analog inhibitor at resolutions of 2.5 and 2.1 Å, respectively. In the crystal structure of the complex, the bound inhibitor is phosphorylated at the sulfoximido nitrogen and is coordinated to three Mg2+ ions. The cysteine-binding site was identified; it is formed inductively at the transition state. In the unliganded structure, an open space exists around the representative cysteine-binding site and is probably responsible foi the competitive binding of glutathione. Upon inhibitor binding, the side chains of Tyr-241 and Tyr-300 turn, forming a hydrogen-bonding triad with the carboxyl group of the inhibitors cysteine moiety, allowing this moiety to fit tightly into the cysteine-binding site with concomitant accommodation of its side chain into a shallow pocket This movement is caused by a conformational change of a switch loop (residues 240-249). Based on this crystal structure, the cysteine-binding sites of mammalian and parasitic 1GCSs were predicted by multiple sequence alignment, although no significant sequence identity exists between the E. coli γGCS and its eukaryotic homologues. The identification of this cysteine-binding site provides important information for the rational design of novel γGCS inhibitors. [ABSTRACT FROM AUTHOR]

Describes the refined crystal structure of the Escherichia coli glutathione synthetase (GSHase) complexed with ADP, GSH, two Mg2+ ions and one sulfate ion bound at the active site. Pseudo enzyme-substrate complex in the reverse reaction; Active site architecture of GSHase; Structure of the quaternary complex of the enzyme in the pseudo-Michaelis complex.

LipB, the lipase activator protein of Pseudomonas aeruginosa TE3285, was overproduced in Escherichia coli, and purified 4.9-fold over the crude extract in the presence of SDS. The purified LipB reactivated the lipase from P. aeruginosa TE3285 denatured with guanidine hydrochloride, and its reactivation did not involve multiple turnover. In this reactivation, a 1: 1 complex between the lipase and LipB was detected in a cross-linking experiment, suggesting that LipB still binds to the lipase after the reactivation. Calcium ion was essential for the complex formation and the reactivation, and addition of EDTA caused inactivation of the reactivated lipase bound to LipB more rapidly than the native lipase. These findings suggest that LipB could affect the calcium binding to the lipase in the reactivation process. LipB was unable to reactivate lipases from other sources except Pseudomonas sp. 109; this lipase has an amino acid sequence which is 98% identical to that of the lipase from P. aeruginosa TE3285. Thus, it may be concluded that LipB specifically recognizes a unique structural element of the lipase. [ABSTRACT FROM AUTHOR]

To elucidate the structure and molecular mechanism of a characteristic proline-specific aminopeptidase produced by the thermophile Aneurinibacillus sp. strain AM-1, its gene was cloned and the recombinant protein was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. X-ray diffraction data were collected to 1.8 Å resolution from the recombinant aminopeptidase crystal. The crystals belong to the orthorhombic space group P21212, with unit-cell parameters a = 93.62, b = 68.20, c = 76.84 Å. A complete data set was also obtained from crystals of SeMet-substituted aminopeptidase. Data in the resolution range 20–2.1 Å from the MAD data set from the SeMet-substituted crystal were used for phase determination. [ABSTRACT FROM AUTHOR]

Escherichia coli B γ-glutamylcysteine synthetase (γOCS) catalyzes the ATP-dependent coupling of L-Glu and L-Cys to form the glutathione precursor γ-L-Glu-Cys and is a target for development of potential therapeutic agents. By introducing four point mutations of surface-exposed cysteine residues to serine, the γOCS was purified to homogeneity; single crystals have been obtained using the hanging-drop vapour-diffusion method with sodium formate. The γOCS crystal diffracted to 2.8 Å and belongs to space group R3, with unit-cell parameters a = b = 326.7, c = 103.9 Å. [ABSTRACT FROM AUTHOR]

Pex19p is a peroxin involved in peroxisomal membrahe biogenesis and probably functions as a chaperone and/or soluble receptor specific for cargo peroxisomal membrane proteins (PMPs). To elucidate the functional constituents of Pex19p in terms of the protein structure, we investigated its domain architecture and binding affinity toward various PMPs and peroxins. The human Pex19p cDNA was overexpressed in Escherichia coli, and a highly purified sample of the Pex19p protein was prepared. When PMP22 was synthesized by cell-free translation in the presence of Pex19p, the PMP22 bound to Pex19p was soluble, whereas PMP22 alone was insoluble. This observation shows that Pex19p plays a role in capturing PMP and maintaining its solubility. In a similar manner, Pex19p was bound to PMP70 and Pex16p as well as the Pex3p soluble fragment. Limited proteolysis analyses revealed that Pexl9p consists of the C-terminal core domain flanking the flexible N-terminal region. Separation of Pex19p into its N- and C-terminal halves abolished interactions with PMP22, PMP70, and Pex16p. In contrast, the flexible N-terminal half of Pex19p was bound to the Pex3p soluble fragment, suggesting that the binding mode of Pex3p toward Pexl9p differs from that of other PMPs. This idea is supported by our detection of the Pex19p-Pex3p-PMP22 ternary complex. [ABSTRACT FROM AUTHOR]