The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (>60% sequence similarity) and methane monooxygenase (>40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly α-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase α-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.     

Determinism of bacterial metacommunity dynamics in the southern East China Sea varies depending on hydrography

Spatial variation of communities composition (metacommunities) results from multiple assembly mechanisms, including environmental filtering and dispersal; however, whether and why the relative importance of the assembly mechanisms in shaping bacterial metacommunity changes through time in marine pelagic systems remains poorly studied. Here, we applied the elements of metacommunity structure framework and the variation partitioning framework to examine whether temporal variation of hydrographic conditions influences bacterioplankton metacommunity dynamics in the southern East China Sea (ECS). The spatiotemporal variation of bacterial communities composition was revealed using 454 pyrosequencing of 16S rDNA. In addition to the whole bacterial community, we analyzed four dominant taxonomic groups (Cyanobacteria, Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria) separately. Our analyses indicate that, considering the whole community level, the determinism of metacommunity structure varied among seasons. When the degree of connectivity was low (December), the metacommunity exhibited random distribution and was explained mainly by the environmental component. However, Clementsian metacommunity was found at intermediate connectivity (May), during which the environmental and spatial predictors were both significant. When connectivity was high (August), a random distribution pattern was found and no significant effect of environmental filtering or dispersal limitation was detected. Nevertheless, when considering different taxonomic groups, the differences in metacommunity dynamics among groups were found. Our results suggest that the driving forces of metacommunity dynamics varied depending on hydrography, as the degrees of environmental heterogeneity and connectivity among habitat patches were determined by circulation pattern. Moreover, mechanisms varied among different taxonomic groups, suggesting that differential dispersal capacity among taxonomic groups should be integrated into community assembly studies.     

Signaling and function of caspase and c-jun N-terminal kinase in cisplatin-induced apoptosis

Caspases and c-Jun N-terminal kinase (JNK) are activated in tumor cells during induction of apoptosis. We investigated the signaling cascade and function of these enzymes in cisplatin-induced apoptosis. Treatment of Jurkat T-cells with cisplatin induced cell death with DNA fragmentation and activation of caspase and JNK. Bcl-2 overexpression suppressed activation of both enzymes, whereas p35 and CrmA inhibited only the DEVDase (caspase-3-like) activity, indicating that the activation of these enzymes may be differentially regulated. Cisplatin induced apoptosis with the cytochrome c release and caspase-3 activation in both wild-type and caspase-8-deficient JB-6 cells, while the Fas antibody induced these apoptotic events only in wild-type cells. This indicates that caspase-8 activation is required for Fas-mediated apoptosis, but not cisplatin-induced cell death. On the other hand, cisplatin induced the JNK activation in both the wild-type and JB-6 cells, and the caspase-3 inhibitor Z-DEVD-fmk did not inhibit this activation. The JNK overexpression resulted in a higher JNK activity, AP-1 DNA binding activity, and metallothionein expression than the empty vector-transfected cells following cisplatin treatment. It also partially protected the cells from cisplatin-induced apoptosis by decreasing DEVDase activity. These data suggest that the cisplatin-induced apoptotic signal is initiated by the caspase-8-independent cytochrome c release, and the JNK activation protects cells from cisplatin-induced apoptosis via the metallothionein expression.     

Microbial processes and temperature in Chesapeake Bay: current relationships and potential impacts of regional warming

The importance of temperature in regulating physiological processes is without question; however, the interpretation of the relationship between temperature and ecological data is much more complicated. Consequently, it is difficult to decide how the nature of the temperature response terms should be included in models used to predict responses of microbial processes to increasing regional temperature. This analysis compiles several years of data from a research programme conducted in Chesapeake Bay, in an effort to examine how individual microbial processes ? as well as the balance between autotrophy and heterotrophy ? have responded to temperature, and to predict changes in microbial trophic state based on realistic increases in global temperature. The upper boundary on all of the pelagic microbial rate processes that were measured could be described remarkably well as a linear function of temperature, although there was substantial scatter in the data. Pelagic microbial rate processes (e.g. phytoplankton production, respiration, bacterial productivity) showed a remarkably constrained range of Q₁₀ values from 1.7 to 3.4. The one notable exception to this was nitrogen uptake in the North and Mid Bay, which exhibited Q₁₀ values < 1.0. Proxies for phytoplankton biomass (e.g. chlorophyll) were largely independent of temperature while bacterial abundance was significantly related to temperature and was found to have a Q₁₀ of 1.88. Using these individual temperature responses, the balance of autotrophy and heterotrophy was assessed by calculating the community photosynthesis to respiration (P:R), NH₄⁺ uptake to regeneration (U:R) and phytoplankton to bacterial productivity (PP:BP) ratios for current conditions (all ratios) and for a 2 and 5 °C temperature increase (NH₄⁺ U:R excluded). The NH₄⁺ U:R ratio stayed remarkable constant at ～1 over the entire temperature range supporting the importance of regenerative processes to nitrogen availability even during periods of heavy allochthonous inputs. These elevated temperature calculations for P:R and PP:BP suggest that the magnitude of autotrophic production during the spring bloom may decrease with increased regional temperature and, as a consequence, the Chesapeake Bay might become net heterotrophic on an annual timescale. These calculations should be considered with caution, but nonetheless demonstrate that the impact of increasing temperature on the balance of autotrophic and heterotrophic processes needs to be researched further.     

Cloning and expression of the genes encoding the propene monooxygenase from Xanthobacter, Py2

Xanthobacter Py2 grows on propene as sole carbon source, converting propene to propene oxide (epoxypropane) using an alkene-specific monooxygenase, as the first step in catabolism. Four mutants, NZ1–4, with a propene^– propene oxide⁺ phenotype were isolated by 1-methyl-3-nitro-1-nitrosoguanidine mutagenesis or by enrichment with the suicide substrate vinylidene chloride, and were shown to have lost the ability to convert alkenes to epoxides. All four mutants were complemented by a number of clones of Xanthobacter Py2 chromosomal DNA in the broad-host-range cosmid pLAFR5, some of which appeared to be non-overlapping. Representatives of the different clones obtained were transferred into Xanthobacter autotrophicus JW33 and one, pNY2, the most frequently isolated clone, was shown to express an inducible, fully functional propene monooxygenase. Subcloning revealed that all four mutants were complemented by a 2.4-kb EcoRI-PstI fragment situated at one end of the cosmid insert. However, activity in X. autotrophicus JW33 could only be expressed from pNY2, containing the complete insert (25 kb), suggesting a large operon or some form of long-range control. pNY2 failed to express in E. coli. In X. autotrophicus JW33 [pNY2] at least three new polypeptides were evident after induction with propene compared with a control carrying only the cosmid pLAFR5.     

Summer profundal hypoxia determines the coupling of methanotrophic production and the pelagic food web in a subtropical reservoir

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The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases and catalyzes aromatic monohydroxylation of benzene, toluene, and phenol

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase alpha-subunit (XamoA); aamB, encoding the 88-residue oxygenase gamma-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase beta-subunit (XamoE); and aamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of sigma54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase alpha-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase from Rhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacter strain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture of o-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studies Xanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of the aam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol.     

ADAMTS13 substrate recognition of von Willebrand factor A2 domain

ADAMTS13 controls the multimeric size of circulating von Willebrand factor (VWF) by cleaving the Tyr1605-Met1606 bond in theA2 domain. To examine substrate recognition, we expressed in bacteria and purified three A2 (VWF76-(1593-1668), VWF115-(1554-1668), VWFA2-(1473-1668)) and one A2-A3 (VWF115-A3-(1554-1874)) domain fragments. Using high pressure liquid chromatography analysis, the initial rates of VWF115 cleavage by ADAMTS13 at different substrate concentrations were determined, and from this the kinetic constants were derived (Km 1.61 microM; kcat 0.14 s(-1)), from which the specificity constant kcat/Km was calculated, 8.70 x 10(4) m(-1) s(-1). Similar values of the specificity constant were obtained for VWF76 and VWF115-A3. To identify residues important for recognition and proteolysis of VWF115, we introduced certain type 2A von Willebrand disease mutations by site-directed mutagenesis. Although most were cleaved normally, one (D1614G) was cleaved approximately 8-fold slower. Mutagenesis of additional charged residues predicted to be in close proximity to Asp1614 on the surface of the A2 domain (R1583A, D1587A, D1614A, E1615A, K1617A, E1638A, E1640A) revealed up to 13-fold reduction in kcat/Km for D1587A, D1614A, E1615A, and K1617A mutants. When introduced into the intact VWFA2 domain, proteolysis of the D1587A, D1614A, and E1615A mutants was also slowed, particularly in the presence of urea. Surface plasmon resonance demonstrated appreciable reduction in binding affinity between ADAMTS13 and VWF115 mutants (KD up to approximately 1.3 microM), compared with VWF115 (KD 20 nM). These results demonstrate an important role for Asp1614 and surrounding charged residues in the binding and cleavage of the VWFA2 domain by ADAMTS13.     

Cloning, Expression, and Site-Directed Mutagenesis of the Propene Monooxygenase Genes from Mycobacterium sp. Strain M156

Propene monooxygenase has been cloned from Mycobacterium sp. strain M156, based on hybridization with the amoABCD genes of Rhodococcus corallinus B276. Sequencing indicated that the mycobacterial enzyme is a member of the binuclear nonheme iron monooxygenase family and, in gene order and sequence, is most similar to that from R. corallinus B-276. Attempts were made to express the pmoABCD operon in Escherichia coli and Mycobacterium smegmatis mc²155. In the former, there appeared to be a problem resolving overlapping reading frames between pmoA and -B and between pmoC and -D, while in the latter, problems were encountered with plasmid instability when the pmoABCD genes were placed under the control of the hsp60 heat shock promoter in the pNBV1 vector. Fortuitously, constructs with the opposite orientation were constitutively expressed at a level sufficient to allow preliminary mutational analysis. Two PMO active-site residues (A94 and V188) were targeted by site-directed mutagenesis to alter their stereoselectivity. The results suggest that changing the volume occupied by the side chain at V188 leads to a systematic alteration in the stereoselectivity of styrene oxidation, presumably by producing different orientations for substrate binding during catalysis. Changing the volume occupied by the side chain at A94 produced a nonsystematic change in stereoselectivity, which may be attributable to the role of this residue in expansion of the binding site during substrate binding. Neither set of mutations changed the enzyme's specificity for epoxidation.