Crystal structure of D-aminoacylase from Alcaligenes faecalis DA1. A novel subset of amidohydrolases and insights into the enzyme mechanism

D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the hydrolysis of N-acyl-D-amino acids. We report here the first D-aminoacylase crystal structure from A. faecalis at 1.5-A resolution. The protein comprises a small beta-barrel, and a catalytic (betaalpha)(8)-barrel with a 63-residue insertion. The enzyme structure shares significant similarity to the alpha/beta-barrel amidohydrolase superfamily, in which the beta-strands in both barrels superimpose well. Unexpectedly, the enzyme binds two zinc ions with widely different affinities, although only the tightly bound zinc ion is required for activity. One zinc ion is coordinated by Cys(96), His(220), and His(250), while the other is loosely chelated by His(67), His(69), and Cys(96). This is the first example of the metal ion coordination by a cysteine residue in the superfamily. Therefore, D-aminoacylase defines a novel subset and is a mononuclear zinc metalloenzyme but containing a binuclear active site. The preferred substrate was modeled into a hydrophobic pocket, revealing the substrate specificity and enzyme catalysis. The 63-residue insertion containing substrate-interacting residues may act as a gate controlling access to the active site, revealing that the substrate binding would induce a closed conformation to sequester the catalysis from solvent.     

Improved statistical methods for hit selection in high-throughput screening

High-throughput screening (HTS) plays a central role in modern drug discovery, allowing the rapid screening of large compound collections against a variety of putative drug targets. HTS is an industrial-scale process, relying on sophisticated automation, control, and state-of-the art detection technologies to organize, test, and measure hundreds of thousands to millions of compounds in nano- to microliter volumes. Despite this high technology, hit selection for HTS is still typically done using simple data analysis and basic statistical methods. The authors discuss in this article some shortcomings of these methods and present alternatives based on modern methods of statistical data analysis. Most important, they describe and show numerous real examples from the biologist-friendly Stat Server HTS application (SHS), a custom-developed software tool built on the commercially available S-PLUS and StatServer statistical analysis and server software. This system remotely processes HTS data using powerful and sophisticated statistical methodology but insulates users from the technical details by outputting results in a variety of readily interpretable graphs and tables.     

Comparison of terbutaline and dobutamine in rats with endotoxemia

It is generally accepted that bacterial endotoxin (lipopolysaccharide, LPS) acts via endogenous mediators leading to endotoxicity. Among these endogenous mediators, tumor necrosis factor-alpha (TNF-alpha) seems to induce all characteristics for endotoxemia. Inhibition of TNF-(alpha production by cAMP-elevating agents has been well documented. Terbutaline (an agonist of beta2-adrenoceptor) and dobutamine (an agonist of beta1-adrenoceptor), both are able to increase intracellular cAMP via activation of adenylate cyclase, were examined in the anesthetized rat with endotoxemia. Terbutaline or dobutamine was administered to the rat at 30 min after LPS injection. Hemodynamic changes and plasma TNF-alpha and nitrate (the end product of nitric oxide [NO]) levels as well as superoxide anion (O2*-) production in the aorta were examined in this study. Results showed that terbutaline, but not dobutamine, improved the circulatory failure (e.g. hypotension and vascular hyporeactivity) in rats with endotoxemia. In addition, both terbutaline and dobutamine reduced the plasma TNF-alpha level, but only terbutaline attenuated the aortic O2*- production in these endotoxemic rats. The beneficial effect of terbutaline in endotoxemic animals was associated with a reduction in plasma TNF-alpha and aortic O2*-, but not in plasma NO.     

Lactococcus garvieae infection in the giant freshwater prawn Macrobranchium rosenbergii confirmed by polymerase chain reaction and 16S rDNA sequencing.

An epizootic bacterial infection in the giant freshwater prawn Macrobranchium rosenbergii occurred in Taiwan from May to June 1999. The cumulative mortality was approximately 30 to 75%. The diseased prawns showed opaque and whitish muscles and were approximately 2 mo old with total lengths from 5 to 6 cm. Histopathologically, they showed marked edema and necrotic lesions with inflammation in the muscles and hepatopancreas. Bacteria isolated using brain heart infusion medium or tryptic soy agar were Gram-positive and ovoid. Three isolates from diseased prawns at different farms were tested using the API 20 Strepsystem and conventional tests and identified as Lactococcus garvieae. Experimental infections with these isolates gave gross signs and histopathological changes similar to those seen in the naturally infected prawns. The LD50 value of isolate MR1 was 6.6 x 10(5) colony forming units/prawn. Identification of MR1 was confirmed by a PCR assay for L. garvieae that gave the expected amplicon of 1100 bp. In addition, its 16S rDNA sequence (GenBank accession number AF283499) gave 99% sequence identity to Enterococcus seriolicida (synonym L. garvieae; GenBank accession number AF061005). This is the first report of confirmed L. garvieae infection in prawn aquaculture.     

Substrate-velocity relationships for the Trichoderma viride cellulase-catalyzed hydrolysis of cellulose.

The influence of substrate and enzyme concentrations on the rate of saccharification of two defined insoluble cellulose substrates, Avicel (FMC Corp., Philadelphia, Pa.) and Solka-Floc (James River Co., Berlin, N.H.), by the cellulase enzyme system of Trichoderma viride was evaluated. In the assays, enzyme concentrations ranging from 0.004 to 0.016 IU/ml and substrate concentrations up to 10% (wt/vol) were used. Analysis by initial velocity methods found the maximum velocity of saccharification to be nearly equivalent for the two substrates and the Km for the two substrates to be of a similar magnitude, i.e., 0.20% (wt/vol) for Solka-Floc and 0.63% (wt/vol) for Avicel. Studies in which relatively high substrate concentrations (greater than 15 times the Km) were used demonstrated that the enzyme exhibited very different apparent substrate inhibition properties for the two substrates. The rate of saccharification of Avicel at relatively high substrate concentrations was up to 35% lower than the maximum rate which was observed at lower substrate concentrations. The Avicel concentration corresponding to the maximum rate of saccharification was dependent on the enzyme concentration. In contrast to the results with Avicel, the enzyme did not exhibit substrate inhibition with the Solka-Floc substrate. Potential differences in the degree of substrate inhibition with different substrates, as reported here, are particularly relevant to the experimental design of comparative studies.     

Crystallization and preliminary X-ray analysis of volvatoxin A2 from Volvariella volvacea

Volvatoxin A2, an ion channel disturbed cardiotoxic and hemolytic protein from the edible mushroom, Volvarilla volvacea, has been crystallized by the vapor diffusion method using polyethylene glycol 4000 and ammonium sulfate in sodium acetate buffer pH 4.6. The best crystals belong to the monoclinic space group C2 with unit cell dimensions a = 155.25 Å, b = 58.06 Å, c = 116.92 Å, and β = 119.5°. These crystals diffract to at least 2.2 Å and there are four molecules of molecular weight 24 kDa per asymmetric unit with a solvent content of 48%.     

Crystal structures of the chromosomal proteins Sso7d/Sac7d bound to DNA containing T-G mismatched base-pairs

Sso7d and Sac7d are two small chromatin proteins from the hyperthermophilic archaeabacterium Sulfolobus solfataricus and Sulfolobus acidocaldarius, respectively. The crystal structures of Sso7d-GTGATCGC, Sac7d-GTGATCGC and Sac7d-GTGATCAC have been determined and refined at 1.45 A, 2.2 A and 2.2 A, respectively, to investigate the DNA binding property of Sso7d/Sac7d in the presence of a T-G mismatch base-pair. Detailed structural analysis revealed that the intercalation site includes the T-G mismatch base-pair and Sso7d/Sac7d bind to that mismatch base-pair in a manner similar to regular DNA. In the Sso7d-GTGATCGC complex, a new inter-strand hydrogen bond between T2O4 and C14N4 is formed and well-order bridging water molecules are found. The results suggest that the less stable DNA stacking site involving a T-G mismatch may be a preferred site for protein side-chain intercalation.     

Systemic infection of Kudoa lutjanus n. sp. (Myxozoa: Myxosporea) in red snapper Lutjanus erythropterus from Taiwan

A new species of Kudoa lutjanus n. sp. (Myxosporea) is described from the brain and internal organs of cultured red snapper Lutjanus erythropterus from Taiwan. The fish, 260 to 390 g in weight, exhibited anorexia and poor appetite and swam in the surface water during outbreaks. Cumulative mortality was about 1% during a period of 3 wk. The red snapper exhibited numerous creamy-white pseudocysts, 0.003 to 0.65 cm (n = 100) in diameter, in the eye, swim bladder, muscle and other internal organs, but especially in the brain. The number of pseudocysts per infected fish was not correlated with fish size or condition. Mature spores were quadrate in apical view and suboval in side view, measuring 8.2 +/- 0.59 microm in width and 7.3 +/- 0.53 microm in length. The 4 valves were equal in size, each with 1 polar capsule. Polar capsules were pyriform in shape, measuring 3.62 +/- 0.49 microm in length and 2.2 +/- 0.49 microm in width. Mild inflammatory responses or liquefaction of host tissue were associated with K. lutjanus n. sp. infection. The junction of shell valves appeared as overlapping, straight lines. The polar filament formed 2 to 3 coils. A general PCR (polymerase chain reaction) primer for Kudoa amplified the small subunit (SSU) rDNA sequences, and the amplified gene was sequenced. It was evident from the phylogenetic tree that the 3 strains tested, AOD93020M, AOD93028M and AOD93028B, were identical and belonged to the Kudoa SS rRNA subgroup. The evolutionary tree showed that these strains form a unique clade, at a distance from other Kudoa species and myxosporeans. The spore's morphological and ultrastructural characteristics, as well as the SS rDNA properties of the isolates, were also essentially identical and served to distinguish them from representative Kudoa. It is, therefore, proposed that the strains isolated from the diseased red snapper be assigned to a new species.     

Identification of a hepatic factor capable of supporting hepatitis C virus replication in a nonpermissive cell line

Although hepatitis C virus E2 protein can bind to human cells by interacting with a putative viral receptor, CD81, the interaction alone is not sufficient to establish permissiveness for hepatitis C virus infection. Using an Epstein-Barr virus-based extrachromosomal replication system, we have screened through a human liver cDNA library and successfully identified a cDNA capable of supporting hepatitis C virus replication in an otherwise nonpermissive cell line. This cDNA encodes a protein exhibiting homology to a group of proteins derived from various evolutionarily distant species, including Oryza sativa submergence-induced protein 2A. The mRNAs encoding this factor are heterogeneous at the 5' ends and are ubiquitously expressed in multiple tissues, albeit in a very small amount. The longest mRNA contains an in-frame and upstream initiation codon and codes for a larger protein. This 5'-extended form of mRNA was detected in hepatocellular carcinoma, but not in normal liver tissue. Immunofluorescence analysis demonstrated that the hepatic factor was distributed evenly in cells, but occasionally formed aggregations in the peri- or intranuclear areas. In summary, we have identified a hepatic factor capable of supporting hepatitis C virus replication in an otherwise nonpermissive cell line. This factor belongs to a previously uncharacterized protein family. The physiological function of this protein awaits further study.     

Mutational analysis of the autoprocessing site of subtilisin YaB-G124A

The potential residue at the autoprocessing site for improving processing efficiency was evaluated from hydrolysis of 19 cleavage-site-mimicking octapeptides, VTTXQTVP (-4 to +4), by the mature subtilisin YaB and YaB-G124A mutants. Both enzymes cleaved the octapeptides mainly at two sites, X-Q (A-site) and Q-T (B-site), at varied preferences. Based on the results above, Met(-1) of YaB-G124A was mutated and, as expected, extracellular enzyme production increased with Gln or Ala replacement, but decreased with Ile or Asp substitution. Together with previous structural studies, our results suggest that autoprocessing is dependent on not only the primary structure, but also the peptide flexibility around the processing site. Cleavage at the B-site resulted in a novel YaB mutant lacking the N-terminus Gln 1, which led the mutant enzyme to less enzymatic activity by 80% and less thermal stability by 20 degrees C, perhaps due to its ligation to the high-affinity calcium ion.