Crystal Structure of Metal-Dependent Allantoinase from Escherichia coli

Allantoinase acts as a key enzyme for the biogenesis and degradation of ureides by catalyzing the conversion of (S)-allantoin into allantoate, the final step in the ureide pathway. Despite limited sequence similarity, biochemical studies of the enzyme suggested that allantoinase belongs to the amidohydrolase family. In this study, the crystal structure of allantoinase from Escherichia coli was determined at 2.1 Å resolution. The enzyme consists of a homotetramer in which each monomer contains two domains: a pseudo-triosephosphate-isomerase barrel and a β-sheet. Analogous to other enzymes in the amidohydrolase family, allantoinase retains a binuclear metal center in the active site, embedded within the barrel fold. Structural analyses demonstrated that the metal ions in the active site ligate one hydroxide and six residues that are conserved among allantoinases from other organisms. Functional analyses showed that the presence of zinc in the metal center is essential for catalysis and enantioselectivity of substrate. Both the metal center and active site residues Asn94 and Ser317 play crucial roles in dictating enzyme activity. These structural and functional features are distinctively different from those of the metal-independent allantoinase, which was very recently identified.     

Enhanced production of recombinant protein inEscherichia coli using silkworm hemolymph

The effect of silkworm hemolymph on the expression of recombinant protein inEscherichia coli was investigated. The addition of silkworm hemolymph to the culture medium increased the production of recombinant β-galactosidase inE. coli. The production was dependent on the concentration of the added silkworm hemolymph, which increased 2-, 5-, and 8-fold in media supplemented with 1,3, and 5% silkworm hemolymph, respectively. To identify the effective component, the silkworm hemolymph was fractionated by gel filtration column chromatography. A fraction, with a molecular weight of about 30 K was identified as the effective component.     

The deubiquitinating enzyme USP37 enhances CHK1 activity to promote the cellular response to replication stress

The deubiquitinating enzyme USP37 is known to contribute to timely onset of S phase and progression of mitosis. However, it is not clear if USP37 is required beyond S-phase entry despite expression and activity of USP37 peaking within S phase. We have utilized flow cytometry and microscopy to analyze populations of replicating cells labeled with thymidine analogs and monitored mitotic entry in synchronized cells to determine that USP37-depleted cells exhibited altered S-phase kinetics. Further analysis revealed that cells depleted of USP37 harbored increased levels of the replication stress and DNA damage markers γH2AX and 53BP1 in response to perturbed replication. Depletion of USP37 also reduced cellular proliferation and led to increased sensitivity to agents that induce replication stress. Underlying the increased sensitivity, we found that the checkpoint kinase 1 is destabilized in the absence of USP37, attenuating its function. We further demonstrated that USP37 deubiquitinates checkpoint kinase 1, promoting its stability. Together, our results establish that USP37 is required beyond S-phase entry to promote the efficiency and fidelity of replication. These data further define the role of USP37 in the regulation of cell proliferation and contribute to an evolving understanding of USP37 as a multifaceted regulator of genome stability.     

Physiological and fermentation properties of <Emphasis Type="Italic">Bacillus coagulans</Emphasis> and a mutant lacking fermentative lactate dehydrogenase activity

Bacillus coagulans, a sporogenic lactic acid bacterium, grows optimally at 50–55°C and produces lactic acid as the primary fermentation product from both hexoses and pentoses. The amount of fungal cellulases required for simultaneous saccharification and fermentation (SSF) at 55°C was previously reported to be three to four times lower than for SSF at the optimum growth temperature for Saccharomyces cerevisiae of 35°C. An ethanologenic B. coagulans is expected to lower the cellulase loading and production cost of cellulosic ethanol due to SSF at 55°C. As a first step towards developing B. coagulans as an ethanologenic microbial biocatalyst, activity of the primary fermentation enzyme L-lactate dehydrogenase was removed by mutation (strain Suy27). Strain Suy27 produced ethanol as the main fermentation product from glucose during growth at pH 7.0 (0.33 g ethanol per g glucose fermented). Pyruvate dehydrogenase (PDH) and alcohol dehydrogenase (ADH) acting in series contributed to about 55% of the ethanol produced by this mutant while pyruvate formate lyase and ADH were responsible for the remainder. Due to the absence of PDH activity in B. coagulans during fermentative growth at pH 5.0, the l-ldh mutant failed to grow anaerobically at pH 5.0. Strain Suy27-13, a derivative of the l-ldh mutant strain Suy27, that produced PDH activity during anaerobic growth at pH 5.0 grew at this pH and also produced ethanol as the fermentation product (0.39 g per g glucose). These results show that construction of an ethanologenic B. coagulans requires optimal expression of PDH activity in addition to the removal of the LDH activity to support growth and ethanol production.     

Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival.

In order to study the physiological role of acetate metabolism in Escherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate, D-lactate, and L-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by the pta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The pta mutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-beta-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.     

Development of an HbA1c-Based Conversion Equation for Estimating Glycated Albumin in a Korean Population with a Wide Range of Glucose Intolerance

Background

Compared to the golden standard glycation index of HbA1c, glycated albumin (GA) has potentials for assessing insulin secretory dysfunction and glycemic fluctuation as well as predicting diabetic vascular complications. However, the reference ranges of GA and a conversion equation need to be clearly defined. We designed this study to determine the reference ranges in patients with normal glucose tolerance (NGT) based on conventional measures of glycemic status and to devise a conversion equation for calculating HbA1c and GA in a Korean population.

Methodology/Principal Findings

In this multicenter, retrospective, cross-sectional study, we recruited antidiabetic drug-naïve patients with available glycemic variables including HbA1c, GA, and fasting plasma glucose regardless of glucose status. For the reference interval of serum GA, 5^th to 95^th percentile value of GA in subjects with NGT was adopted. The conversion equation between HbA1c and GA was devised using an estimating regression model with unknown break-points method. The reference range for GA was 9.0–14.0% in 2043 subjects. The 95^th percentile responding values for FPG, and HbA1c were approximately 5.49 mmol/l, and 5.6%, respectively. The significant glycemic turning points were 5.868% HbA1c and 12.2% GA. The proposed conversion equation for below and above the turning point were GA (%) = 6.960+0.8963 × HbA1c (%) and GA (%) = −9.609+3.720 × HbA1c (%), respectively.

Conclusions/Significance

These results should be helpful in future studies on the clinical implications of high GA relative to HbA1c and the clinical implementation of diabetes management.     

CAS agar diffusion assay for the measurement of siderophores in biological fluids

We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.     

Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha

The actin gene (ACT) from the methylotrophic yeast Hansenula polymorpha was cloned and its structural feature was characterized. In contrast to the actin genes of other ascomycetous yeasts, which have only one large intron, the H. polymorpha ACT gene was found to be split by two introns. The H. polymorpha ACT introns were correctly processed in the heterologous host Saccharomyces cerevisiae despite appreciable differences in the splice site sequences. The promoter region of H. polymorpha ACT displayed two CCAAT motifs and two TATA-like sequences in a configuration similar to that observed in the S. cerevisiae actin promoter. A set of deleted H. polymorpha ACT promoters was exploited to direct expression of the bacterial hygromycin B resistance (hph) gene as a dominant selectable marker in the transformation of H. polymorpha. The resistance level of H. polymorpha transformants to the antibiotic was shown to be dependent on the integration copy number of the hph cassette. The selectivity of the hygromycin B resistance marker for transformants of higher copy number was remarkably increased with the deletion of the upstream TATA-like sequence, but not with the removal of either CCAAT motif, from the H. polymorpha promoter. The dosage-dependent selection system developed in this study should be useful for genetic manipulation of H. polymorpha as an industrial strain to produce recombinant proteins.     

Extracts ofPhellinus gilvus andPhellinus baumii inhibit pulmonary inflammation induced by lipopolysaccharide in rats

Compared to saline-challenged rats, rats exposed to 50 microg intratracheal lipopolysaccharide showed an increase of total white cells (from 0.3 x 10(6) to 2.4 x 10(6)), neutrophils (from 0.09 x 10(6) to 1.8 x 10(6)), the levels of tumor necrosis factor (TNF)-alpha (from 200 pg ml(-1) to 1200 pg ml(-1)), and interleukin (IL)-1beta (from 220 pg ml(-1) to 650 pg ml(-1)) in the bronchial lavage fluid. However, after pretreatment with extracts of Phellinus gilvus and Phellinus baumii, the total white cells, neutrophils, and the level of IL-1beta in lipopolysaccharide-challenged rats were similar to those in saline-challenged rats, except for TNF-alpha. The results indicate that extracts of P. gilvus and P. baumii may be useful in preventing acute pulmonary inflammation in human diseases.