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Urinary-tract infections (UTIs), which are some of the most common infectious diseases in humans, can cause sepsis and death without proper treatment. Therefore, it is necessary to understand their pathogenicity for proper diagnosis and therapeutics. UropathogenicEscherichia coli, the major causative agents of UTIs, contain several genes that are absent in nonpathogenic strains and are therefore considered to be relevant to UTI pathogenicity. c4763 is one of the uropathogenicE. coli-specific proteins, but its function is unknown. To investigate the function of c4763 and its possible role in UTI pathogenicity, its crystal structure was determined at a resolution of 1.45 Å by a multiple-wavelength anomalous diffraction method. c4763 is a homodimer with 129 residues in one subunit that contains a GGCT-like domain with five α-helices and seven β-strands. c4763 shows structural similarity to the C-terminal domain of allophanate hydrolase fromKluyveromyces lactis, which is involved in the degradation of urea. These results suggest that c4763 might be involved in the utilization of urea, which is necessary for bacterial survival in the urinary tract. Further biochemical and physiological investigation will elucidate its functional relevance in UTIs.

Bacterial pathogens have evolved diverse types of efficient machinery to acquire haem, the most abundant source of iron in the human body, and degrade it for the utilization of iron. Gram-positive bacteria commonly encode IsdG-family proteins as haem-degrading monooxygenases.Listeria monocytogenesis predicted to possess an IsdG-type protein (Lmo2213), but the residues involved in haem monooxygenase activity are not well conserved and there is an extra N-terminal domain in Lmo2213. Therefore, its function and mechanism of action cannot be predicted. In this study, the crystal structure of Lmo2213 was determined at 1.75 Å resolution and its haem-binding and haem-degradation activities were confirmed. Structure-based mutational and functional assays of this protein, designated as an Isd-typeL. monocytogeneshaem-degrading enzyme (Isd-LmHde), identified that Glu71, Tyr87 and Trp129 play important roles in haem degradation and that the N-terminal domain is also critical for its haem-degrading activity. The haem-degradation product of Isd-LmHde is verified to be biliverdin, which is also known to be the degradation product of other bacterial haem oxygenases. This study, the first structural and functional report of the haem-degradation system inL. monocytogenes, sheds light on the concealed haem-utilization system in this life-threatening human pathogen.

The structures and reaction mechanisms of enantioselective hydrolases, which can be used in industrial applications such as biotransformations, are largely unknown. Here, the X-ray crystallographic study of a novel (S)-specific esterase (pfEstA) fromPseudomonas fluorescensKCTC 1767, which can be used in the production of (S)-ketoprofen, is described. Multiple sequence alignments with other hydrolases revealed thatpfEstA contains a conserved Ser67 within the S-X-X-K motif as well as a highly conserved Tyr156. Recombinant protein containing an N-terminal His tag was expressed inEscherichia coli, purified to homogeneity and characterized using SDS–PAGE, MALDI-TOF MS and enantioselective analysis.pfEstA was crystallized using a solution consisting of 1 Msodium citrate, 0.1 MCHES pH 9.5, and X-ray diffraction data were collected to a resolution of 1.9 Å with anRmergeof 7.9%. The crystals ofpfEstA belonged to space groupP212121, with unit-cell parametersa= 65.31,b= 82.13,c = 100.41 Å, α = β = γ = 90°.

Peptide deformylase (PDF) catalyzes the removal of the formyl group from the N-terminal methionine residue in newly synthesized polypeptides, which is an essential process in bacteria. Four new inhibitors of PDF that belong to two different classes, hydroxamate/pseudopeptide compounds [PMT387 (7a) and PMT497] and reverse-hydroxamate/nonpeptide compounds [PMT1039 (15e) and PMT1067], have been developed. These compounds inhibited the growth of several pathogens involved in respiratory-tract infections, such as Streptococcus pneumoniae, Moraxella catarrhalis and Haemophilus influenzae, and leading nosocomial pathogens such as Staphylococcus aureus and Klebsiella pneumoniae with a minimum inhibitory concentration (MIC) in the range 0.1-0.8 mg ml(-1). Interestingly, the reverse-hydroxamate/nonpeptide compounds showed a 250-fold higher antimicrobial activity towards S. aureus, although the four compounds showed similar K-i values against S. aureus PDF enzymes, with Ki values in the 11-85 nM range. To provide a structural basis for the discovery of additional PDF inhibitors, the crystal structures of S. aureus PDF in complex with the four inhibitors were determined at resolutions of 1.90-2.30 angstrom. The inhibitor-bound structures displayed distinct deviations depending on the inhibitor class. The distance between the Zn2+ ion and the carbonyl O atom of the hydroxamate inhibitors (or the hydroxyl O atom of the reverse-hydroxamate inhibitors) appears to be correlated to S. aureus inhibition activity. The structural information reported in this study should aid in the discovery of new PDF inhibitors that can be used as novel antibacterial drugs.

Industrial demand for active biocatalysts with desirable biochemical properties is constantly increasing and the discovery and characterization of novel esterases is potentially useful for industrial processes. Here, X-ray crystallographic studies of an (R)-specific SGNH arylesterase (Sm23) from Sinorhizobium meliloti 1021 are reported. The recombinant protein was expressed in Escherichia coli with a His tag and purified to homogeneity. Sm23 was crystallized using 0.2 M magnesium formate as a precipitant and X-ray diffraction data were collected to a resolution of 2.2 Å with an R(merge) of 6.9%. The crystals of SM23 belonged to the I-centred tetragonal space group I4(1)22, with unit-cell parameters a = b = 126.6, c = 190.9 Å. A molecular-replacement solution was obtained using the crystal structure of arylesterase from Mycobacterium smegmatis as a template.

beta-Lactam antibiotics such as penicillins and cephalosporins have a four-atom ring as a common element in their structure. The beta-lactamases, which catalyze the inactivation of these antibiotics, are of great interest because of their high incidence in pathogenic bacteria. A novel oligomeric class C beta-lactamase (Est-Y29) from a metagenomic library was expressed, purified and crystallized. The recombinant protein was expressed in Escherichia coli with an N-terminal 6xHis tag and purified to homogeneity. EstY-29 was crystallized and X-ray intensity data were collected to 1.49 A resolution using synchrotron radiation.

SdiA enhances cell division by regulating the ftsQAZ operon in Escherichia coli as a transcription activator. In addition, SdiA is suggested to play a role in detecting quorum signals that emanate from other species. It is therefore a homologue of LuxR, a cognate quorum-sensing receptor that recognizes a quorum signal and activates the quorum responses. To elucidate the role of SdiA and its functional and structural relationship to LuxR, structural studies were performed on E. coli SdiA. Recombinant SdiA was overexpressed, purified and crystallized at 287 K using the hanging-drop vapour-diffusion method. X-ray diffraction data from a native crystal were collected with 99.7% completeness to 2.7 A resolution with an R(merge) of 6.0%. The crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 130.47, c = 125.23 A.

UDP-glucose pyrophosphorylase (UGPase) catalyzes the synthesis of UDP-glucose, an essential metabolite in all living organisms. An X-ray crystallographic study of UGPase from Helicobacter pylori has been performed in order to elucidate its role in the regulation of this important metabolic pathway. UGPase was crystallized from 0.1 M sodium acetate trihydrate pH 4.6, 2.0 M ammonium sulfate and 0.1 M guanidine-HCl. According to diffraction data collected at a resolution of 2.9 A using a synchrotron-radiation source, the crystal belongs to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 91.47, b = 98.61, c = 245.70 A, alpha = beta = gamma = 90.0 degrees.

ClpX, a member of the HSP (heat-shock protein) 100 family, functions as a molecular chaperone and is a regulatory subunit of the ClpXP protease. To understand the chaperone and regulatory mechanisms of ClpX, Helicobacter pylori ClpX has been overexpressed in Escherichia coli and crystallized at 295 K using (NH(4))(2)HPO(4) as precipitant. X-ray diffraction data have been collected to 2.6 A resolution using a synchrotron-radiation source. The crystals belong to the hexagonal space group P6(5) or P6(1), with unit-cell parameters a = b = 78.52 (04), c = 131.51 (09) A, alpha = beta = 90, gamma = 120 degrees. The crystallographic asymmetric unit contains one molecule of ClpX, with a corresponding V(M) of 2.78 A(3) Da(-1) and a solvent content of 55.8%.

Ygr203p, a 148-residue protein encoded by the ygr203w gene of Saccharomyces cerevisiae, is a homologue of the yeast Acr2 arsenate reductase encoded by the acr2 (or ypr200c) gene. It also shows significant sequence similarity to the human cell-cycle control Cdc25 phosphatase family. It has been overexpressed in soluble form in Escherichia coli with a His(6) tag at its C-terminus. The recombinant protein has been crystallized at 296 K using sodium chloride as precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 40.48, b = 50.95, c = 91.95 A. The asymmetric unit contains a monomer, giving a crystal volume per protein mass (V(m)) of 2.61 A(3) Da(-1) and a solvent content of 53.8%. The crystals diffract to better than 1.9 A resolution with Cu Kalpha X-rays. They are therefore suitable for high-resolution structure determination.

The author list in Duonget al.[(2014).Acta Cryst.D70, 615–626] is corrected.