Acta Crystallographica Section F-structural Biology and Crystallization Communications最新文献

Inosine 5'-monophosphate dehydrogenase (IMPDH) represents a potential antimicrobial drug target. The crystal structure of recombinant Pseudomonas aeruginosa IMPDH has been determined to a resolution of 2.25 Å. The structure is a homotetramer of subunits dominated by a (β/α)8-barrel fold, consistent with other known structures of IMPDH. Also in common with previous work, the cystathionine β-synthase domains, residues 92-204, are not present in the model owing to disorder. However, unlike the majority of available structures, clearly defined electron density exists for a loop that creates part of the active site. This loop, composed of residues 297-315, links α8 and β9 and carries the catalytic Cys304. P. aeruginosa IMPDH shares a high level of sequence identity with bacterial and protozoan homologues, with residues involved in binding substrate and the NAD+ cofactor being conserved. Specific differences that have been proven to contribute to selectivity against the human enzyme in a study of Cryptosporidium parvum IMPDH are also conserved, highlighting the potential value of IMPDH as a drug target.

High-throughput imaging of protein crystallization experiments with ultraviolet (UV) light has recently become commercially available and can enable crystallographers to differentiate between crystals of protein and those of salt, as the visualization of protein crystals is based on intrinsic tryptophan fluorescence. Unfortunately, UV imaging is not a panacea, as some protein crystals will not fluoresce under UV excitation and some salt crystals are UV-fluorescently active. As a new technology, there is little experience within the general community on how to use this technology effectively and what caveats to look out for. Here, an attempt is made to identify some of the common problems that may arise using UV-imaging technology by examining test proteins, common crystallization reagents and a range of proteins by assessing their UV-Vis absorbance spectra. Some pointers are offered as to which systems may not be appropriate for this methodology.

The major means of horizontal gene spread (e.g. of antibiotic resistance) is conjugative plasmid transfer. It presents a serious threat especially for hospitalized and immuno-suppressed patients, as it can lead to the accelerated spread of bacteria with multiple antibiotic resistances. Detailed information about the process is available only for bacteria of Gram-negative (G-) origin and little is known about the corresponding mechanisms in Gram-positive (G+) bacteria. Here we present the purification, biophysical characterization, crystallization and preliminary structure determination of the TraM C-terminal domain (TraMΔ, comprising residues 190-322 of the full-length protein), a putative transfer protein from the G+ conjugative model plasmid pIP501. The crystals diffracted to 2.5 Å resolution and belonged to space group P1, with unit-cell parameters a = 39.21, b = 54.98, c = 93.47 Å, α = 89.91, β = 86.44, γ = 78.63° and six molecules per asymmetric unit. The preliminary structure was solved by selenomethionine single-wavelength anomalous diffraction.

Bacterial infections are increasingly difficult to treat owing to the spread of antibiotic resistance. A major concern is Gram-negative bacteria, for which the discovery of new antimicrobial drugs has been particularly scarce. In an effort to accelerate early steps in drug discovery, the EU-funded AEROPATH project aims to identify novel targets in the opportunistic pathogen Pseudomonas aeruginosa by applying a multidisciplinary approach encompassing target validation, structural characterization, assay development and hit identification from small-molecule libraries. Here, the strategies used for target selection are described and progress in protein production and structure analysis is reported. Of the 102 selected targets, 84 could be produced in soluble form and the de novo structures of 39 proteins have been determined. The crystal structures of eight of these targets, ranging from hypothetical unknown proteins to metabolic enzymes from different functional classes (PA1645, PA1648, PA2169, PA3770, PA4098, PA4485, PA4992 and PA5259), are reported here. The structural information is expected to provide a firm basis for the improvement of hit compounds identified from fragment-based and high-throughput screening campaigns.

Sperm whale myoglobin (Mb) has weak dehaloperoxidase activity and catalyzes the peroxidative dehalogenation of 2,4,6-trichlorophenol (TCP) to 2,6-dichloroquinone. Crystals of Mb and of its more active G65T variant were used to study the binding of TCP, 4-iodophenol (4-IP) and phenol. The structures of crystals soaked overnight in a 10 mM solution of phenol revealed that a phenol molecule binds in the proximal cavity, forming a hydrogen bond to the hydroxyl of Tyr146 and hydrophobic contacts which include interactions with Cβ and Cγ of the proximal histidine His93. The phenol position corresponds to the strongest xenon binding site, Xe1. It appears that the ligand enters the proximal cavity through a gate formed by the flexible loops 79-86 and 93-103. TCP and 4-IP do not bind to Mb in this manner under similar conditions; however, it appears to be likely that dimethyl sulfoxide (DMSO), which was used at a concentration of 0.8 M to facilitate 4-IP dissolution, binds in the phenol/Xe1 binding site. In this structure, a water molecule coordinated to the heme iron was replaced by an oxygen molecule, reflecting the reduction of the heme. Crystals of Mb and G65T Mb soaked for 5-10 min did not show bound phenol. Kinetic studies of TCP dechlorination showed that phenol has a dual effect: it acts as a competitive inhibitor that is likely to interfere with TCP binding at the heme edge and as a weak activator, likely through binding in the proximal cavity. The lack of phenol bound at the heme edge in the crystal structures suggests that its inhibitory binding only takes place when the heme is activated by hydrogen peroxide.

Crystal structures of the hexanucleotide d(CACGCG)·d(CGCGTG) were determined in two crystal lattices when different concentrations of the counterion Mn2+ were used in crystallization. The availability of Mn2+ during the crystallization process appears to play an important role in inducing different crystal packings that lead to crystals belonging to the two space groups P2(1) and P6(5). Analysis of the molecular interactions of Mn2+ with the Z-form duplexes shows direct coordination to the purine residues G and A.

Hydrogen sulfide produced by oral bacteria is responsible for oral malodour. Two homologous hydrogen sulfide-producing enzymes, Fn1220 and Cdl, from Fusobacterium nucleatum (which actively produces hydrogen sulfide) were overproduced, purified and crystallized. X-ray diffraction data were collected from the crystals using a synchrotron-radiation source. The Fn1220 crystal belonged to tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 (unit-cell parameters a=b=116.8, c=99.2 Å) and the Cdl crystal belonged to monoclinic space group P2(1) (unit-cell parameters a=84.9, b=70.9, c=87.6 Å, β=90.3°).

The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a dipyrromethane cofactor which is covalently linked by a thioether bridge to an invariant cysteine residue. Since PBGD catalyses a reaction which is common to the biosynthesis of both haem and chlorophyll, structural studies of a plant PBGD enzyme offer great potential for the discovery of novel herbicides. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. Expression in E. coli of a codon-optimized gene for Arabidopsis thaliana PBGD has permitted for the first time the crystallization and preliminary X-ray analysis of the enzyme from a plant species at high resolution.