Mechanistic kinetic model for symmetric carboligations using benzaldehyde lyase

For reactions using thiamine diphosphate (ThDP)-dependent enzymes many empirically-derived kinetic models exist. However, there is a lack of mechanistic kinetic models. This is especially true for the synthesis of symmetric 2-hydroxy ketones from two identical aldehydes, with one substrate acting as the donor and the other as the acceptor. In this contribution, a systematic approach for deriving such a kinetic model for thiamine diphosphate (ThDP)-dependent enzymes is presented. The derived mechanistic kinetic model takes this donor-acceptor principle into account by containing two K(m)-values even for identical substrate molecules. As example the stereoselective carbon-carbon coupling of two 3,5-dimethoxy-benzaldehyde molecules to (R)-3,3',5,5'-tetramethoxy-benzoin using benzaldehyde lyase (EC 4.1.2.38) from Pseudomonas fluorescens is studied. The model is derived using a model-based experimental analysis method which includes parameter estimation, model analysis, optimal experimental design, in silico experiments, sensitivity analysis and model revision. It is shown that this approach leads to a robust kinetic model with accurate parameter estimates and an excellent prediction capability.     

Broad-host-range Yersinia phage PY100: genome sequence, proteome analysis of virions, and DNA packaging strategy

PY100 is a lytic bacteriophage with a broad host range within the genus Yersinia. The phage forms plaques on strains of the three human pathogenic species Yersinia enterocolitica, Y. pseudotuberculosis, and Y. pestis at 37°C. PY100 was isolated from farm manure and intended to be used in phage therapy trials. PY100 has an icosahedral capsid containing double-stranded DNA and a contractile tail. The genome consists of 50,291 bp and is predicted to contain 93 open reading frames (ORFs). PY100 gene products were found to be homologous to the capsid proteins and proteins involved in DNA metabolism of the enterobacterial phage T1; PY100 tail proteins possess homologies to putative tail proteins of phage AaΦ23 of Actinobacillus actinomycetemcomitans. In a proteome analysis of virion particles, 15 proteins of the head and tail structures were identified by mass spectrometry. The putative gene product of ORF2 of PY100 shows significant homology to the gene 3 product (small terminase subunit) of Salmonella phage P22 that is involved in packaging of the concatemeric phage DNA. The packaging mechanism of PY100 was analyzed by hybridization and sequence analysis of DNA isolated from virion particles. Newly replicated PY100 DNA is cut initially at a pac recognition site, which is located in the coding region of ORF2.     

Lack of Association between the Tagging SNP A+930→G of SOCS3 and Type 2 Diabetes Mellitus: Meta-Analysis of Four Independent Study Populations

Background

The suppressor of cytokine signalling 3 (SOCS3) provides a link between cytokine action and their negative consequences on insulin signalling. Thus SOCS3 is a potential candidate gene for type 2 diabetes (T2DM).

Methodology/Principal Findings

Based on HapMap we identified the polymorphism A+930→G (rs4969168) as a haplotype tagging SNP (htSNP) sufficiently covering the genetic variation of the whole gene. We therefore examined the association between rs4969168 within SOCS3 and T2DM in three independent study populations; one prospective case-cohort study and two cross-sectional study populations. Due to the low frequency of individuals being homozygous for the polymorphism a dominant model of inheritance was assumed. The case-cohort study with 2,957 individuals (764 of them with incident T2DM) showed no effect of the polymorphism on diabetes risk (hazard ratio (95%CI): 0.86 (0.66–1.13); p = 0.3). Within the MeSyBePo-study population 325 subjects had T2DM from a total of 1,897 individuals, while the second cross-sectional cohort included 851 cases of T2DM within a total of 1653 subjects. According to the results in the prospective study, no association with T2DM was found (odds ratio (95%CI): 0.78 (0.54–1.12) for MesyBepo and 1.13 (0.90–1.42) for the Leipzig study population). There was also no association with metabolic subtraits such as insulin sensitivity (p = 0.7), insulin secretion (p = 0.8) or the hyperbolic relation of both, the disposition index (p = 0.7). In addition, no evidence for interaction with BMI or sex was found. We subsequently performed a meta-analysis, additionally including the publicly available data from the T2DM-subcohort of the WTCCC (n = 4,855). The overall odds ratio within that meta-analysis was 0.96 (0.88–1.06).

Conclusions/Significance

There is no strong effect of the common genetic variation within the SOCS3 gene on the development of T2DM.     

mtDNA nt13708A variant increases the risk of multiple sclerosis

Background

Mitochondrial DNA (mtDNA) polymorphism is a possible factor contributing to the maternal parent-of-origin effect in multiple sclerosis (MS) susceptibility.

Methods and Findings

In order to investigate the role of mtDNA variations in MS, we investigated six European MS case-control cohorts comprising >5,000 individuals. Three well matched cohorts were genotyped with seven common, potentially functional mtDNA single nucleotide polymorphisms (SNPs). A SNP, nt13708 G/A, was significantly associated with MS susceptibility in all three cohorts. The nt13708A allele was associated with an increased risk of MS (OR = 1.71, 95% CI 1.28–2.26, P = 0.0002). Subsequent sequencing of the mtDNA of 50 individuals revealed that the nt13708 itself, rather than SNPs linked to it, was responsible for the association. However, the association of nt13708 G/A with MS was not significant in MS cohorts which were not well case-control matched, indicating that the significance of association was affected by the population structure of controls.

Conclusions

Taken together, our finding identified the nt13708A variant as a susceptibility allele to MS, which could contribute to defining the role of the mitochondrial genome in MS pathogenesis.     

High-level production of human collagen prolyl 4-hydroxylase in Escherichia coli.

The collagen prolyl 4-hydroxylases (C-P4Hs), enzymes residing within the lumen of the endoplasmic reticulum, play a central role in the synthesis of all collagens. The vertebrate enzymes are alpha(2)beta(2) tetramers in which the two catalytic sites are located in the alpha subunits, and protein disulfide isomerase serves as the beta subunit. All attempts to assemble an active C-P4H tetramer from its subunits in in vitro cell-free systems have been unsuccessful, but assembly of a recombinant enzyme has been reported in several cell types by coexpression of the two types of subunit. An active type I C-P4H tetramer was obtained here by periplasmic expression in Escherichia coli strains BL21 and RB791. Further optimization for production by stepwise regulated coexpression of its subunits in the cytoplasm of a thioredoxin reductase and glutathione reductase mutant E. coli strain resulted in large amounts of human type I C-P4H tetramer. The specific activity of the C-P4H tetramer purified from the cytoplasmic expression was within the range of values reported for human type I C-P4H isolated as a nonrecombinant enzyme or produced in the endoplasmic reticulum of insect cells, but the expression level, about 25 mg/l in a fermenter, is about 5-10 times that obtained in insect cells. The enzyme expressed in E. coli differed from those present in vivo and those produced in other hosts in that it lacked the N glycosylation of its alpha subunits, which may be advantageous in crystallization experiments.     

Relationship Between Microhabitat Structure and Distribution of Mouse Lemurs (Microcebus spp.) in Northwestern Madagascar

Understanding processes affecting the distribution and abundance of organisms is a central issue in ecology and conservation biology. In northwestern Madagascar, we found an uneven distribution pattern of the golden-brown mouse lemur (Microcebus ravelobensis) and the grey mouse lemur (M. murinus). In one area (JBA) the two species lived sympatrically, whereas in another forest area (JBB) Microcebus ravelobensis occurred exclusively. To investigate whether differences in forest structure may explain this uneven distribution, we conducted a microhabitat analysis and related it to specific distribution. In JBA the habitat of Microcebus ravelobensis was characterized by a higher percentage of trees with many lianas and a higher cover of the herb layer, whereas that of M. murinus had a higher number of trees with a diameter at breast height (DBH) >10 cm. The comparison of the forest structure of the microhabitats of the two species between JBA and JBB revealed further differences. The cover of the overstory, the percentage of trees without lianas and the number of larger trees (DBH >10 cm) among the microhabitats were higher for Microcebus murinus in JBA than for M. ravelobensis in JBB whereas the microhabitats of M. ravelobensis at the two sites did not differ concerning these vegetation structures. Differences between the two species coincide with those of resources important for survival. Our results indicate the importance of microhabitat analyses for the understanding of distribution patterns of species and for successful conservation planning.