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91.
Jan Hendrik Wübbeler Matthias Raberg Ulrike Brandt Alexander Steinbüchel 《Applied and environmental microbiology》2010,76(21):7023-7028
The catabolism of the disulfide 3,3′-dithiodipropionic acid (DTDP) is initiated by the reduction of its disulfide bond. Three independent Tn5::mob-induced mutants of Advenella mimigardefordensis strain DPN7T were isolated that had lost the ability to utilize DTDP as the sole source of carbon and energy and that harbored the transposon insertions in three different sites of the same dihydrolipoamide dehydrogenase gene encoding the E3 subunit of the pyruvate dehydrogenase multi-enzyme complex of this bacterium (LpdAAm). LpdAAm was analyzed in silico and compared to homologous proteins, thereby revealing high similarities to the orthologue in Ralstonia eutropha H16 (PdhLRe). Both bacteria are able to cleave DTDP into two molecules of 3-mercaptopropionic acid (3MP). A. mimigardefordensis DPN7T converted 3MP to 3-sulfinopropionic acid, whereas R. eutropha H16 showed no growth with DTDP as the sole carbon source but was instead capable of synthesizing heteropolythioesters using the resulting cleavage product 3MP. Subsequently, the genes lpdAAm and pdhLRe were cloned, heterologously expressed in Escherichia coli applying the pET23a expression system, purified, and assayed by monitoring the oxidation of NADH. The physiological substrate lipoamide was reduced to dihydrolipoamide with specific activities of 1,833 mkat/kg of protein (LpdAAm) or 1,667 mkat/kg of protein (PdhLRe). Reduction of DTDP was also unequivocally detected with the purified enzymes, although the specific enzyme activities were much lower: 0.7 and 0.5 mkat/kg protein, respectively.In Advenella mimigardefordensis strain DPN7T (15, 42), three independent mutants with an insertion of Tn5::mob in the lpdA gene coding for the E3 component of the pyruvate dehydrogenase multi-enzyme complex revealed an interesting phenotype: these mutants were fully impaired in utilizing 3,3′-dithiodipropionic acid (DTDP) as the sole carbon and energy source, whereas the growth on no other tested carbon sources was affected (41). Our main interest in the catabolism of DTDP is to unravel the pathway and to identify the involved enzymes. Furthermore, the application of this disulfide as precursor substrate for biotechnological production of polythioesters (PTE) (22) is of interest. Since poly(3-mercaptopropionate) (PMP) biosynthesis depends hitherto on supplying the harmful thiol 3-mercaptopropionic acid (3MP) (35), an improvement of the recombinant Escherichia coli system by heterologous expression of enzymes capable of cleaving the less toxic DTDP symmetrically into two molecules of 3MP, which are then polymerized, could be an important achievement toward large-scale biotechnological production of PMP.Two different enzyme systems catalyzing the conversion of disulfides into the corresponding thiols are already known and have been described in detail. (i) Enzymes belonging to the well-characterized family of pyridine-nucleotide disulfide oxidoreductases (25) contain a redox center formed by a disulfide bridge coupled to a flavin ring. They catalyze a simultaneous two-electron transfer via the enzymatic active disulfides associated with the pyridine nucleotides and flavin, toward the substrate (39, 40). (ii) An alternative disulfide reduction is catalyzed by enzymes using iron-sulfur clusters to cleave of disulfide substrates in two one-electron reduction steps (37). The disrupted gene in A. mimigardefordensis was designated lpdAAm (EC 1.8.1.4), and it encodes a homodimeric flavoprotein, the dihydrolipoamide dehydrogenase LpdAAm (i.e., the E3 component of the pyruvate dehydrogenase multi-enzyme complex of A. mimigardefordensis strain DPN7T) belonging to the above-mentioned family of pyridine nucleotide-disulfide oxidoreductases. Enzymes of this class share high sequence and structural similarities and catalyze reduction of compounds which are linked by disulfide bonds (38). Alkylhydroperoxide reductases, coenzyme A disulfide reductases, glutathione reductases, mycothione reductases, thioredoxin reductases, and trypanothione reductases also, in addition to dihydrolipoamide dehydrogenases, belong to this family (3, 38). The physiological function of LpdAAm is most probably the conversion of lipoamide to dihydrolipoamide, but the reduction of DTDP into two molecules of 3MP (Fig. (Fig.1)1) is also predicted, enabling the first step in DTDP catabolism in A. mimigardefordensis strain DPN7T (41).Open in a separate windowFIG. 1.Reactions catalyzed by LpdAAm and PdhLRe. Presented are the enzymatic conversions of DTDP into two molecules of 3MP (A), lipoamide into dihydrolipoamide (B), and DTNB into two molecules of NTB (C). Abbreviations: DTDP, 3,3′-dithiodipropionic acid; 3MP, 3-mercaptopropionic acid; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); NTB, 2-nitro-5-thiobenzoic acid.Ralstonia eutropha H16 synthesizes copolymers of 3-hydroxybutyrate and 3MP, if 3MP (23) or DTDP (22) is supplied as a precursor in addition to a second utilizable carbon source. Although R. eutropha is not able to grow with DTDP as the sole carbon source, it must be capable of cleaving this organic disulfide symmetrically, because it synthesizes from it heteropolymers containing the resulting 3MP. Thus, R. eutropha must possess at least one gene encoding a DTDP-cleaving enzyme. Five genes coding for homologues of a dihydrolipoamide dehydrogenase (DHLDH), which in A. mimigardefordensis DPN7T is obviously involved in DTDP degradation, are known to exist in the genome of R. eutropha H16 (27; M. Raberg, J. Bechmann, U. Brandt, J. Schlüter, B. Uischner, and A. Steinbüchel, unpublished data). Therefore, LpdAAm and the five DHLDH paralogues of R. eutropha H16 were aligned and compared (Fig. (Fig.2).2). Subsequently, lpdAAm and the gene encoding the DHLDH belonging to the pyruvate dehydrogenase complex of R. eutropha H16 (pdhLRe) were cloned, heterologously expressed in Escherichia coli, purified, and assayed.Open in a separate windowFIG. 2.Phylogenetic relationships of the A. mimigardefordensis strain DPN7T LpdA (boldface), R. eutropha H16 PdhL (boldface), and homologues. The neighbor-joining plot was derived from a CLUSTAL X alignment of amino acid sequences closely related to LpdAAm. The amino acid sequence of the outer membrane protein P64K from Neisseria meningitidis was used as the outgroup. GenBank accession numbers are given in parentheses. Scale bar, 10% sequence divergence. 相似文献
92.
An immunoaffinity liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantitation of the zinc endopeptidase matrix metalloproteinase 9 (MMP-9) from mouse serum. Sample preparation for the assay included magnetic bead-based enrichment using an MMP-9 antibody and was performed in a 96-well plate format using a liquid-handling robotic platform. The surrogate peptide GSPLQGPFLTAR derived from MMP-9 by trypsin digestion was monitored using an on-line capillary flow trap-release chromatography setup incorporating a series of trap columns (C18, strong cation exchange, and another C18) prior to nanoflow chromatography and nanospray ionization with selected reaction monitoring (SRM) detection. The assay was fit-for-purpose validated and found to be accurate (<15% interbatch relative error) and precise (<15% interbatch coefficient of variation) across a range from 0.03 to 7.3 nM mouse MMP-9. Finally, the method was employed to measure MMP-9 concentrations in 30 naïve mouse serum samples, and results were compared with those obtained by an immunoassay. 相似文献
93.
94.
Melissa R. van Dijk Ben C. L. van Schaijk Shahid M. Khan Maaike W. van Dooren Jai Ramesar Szymon Kaczanowski Geert-Jan van Gemert Hans Kroeze Hendrik G. Stunnenberg Wijnand M. Eling Robert W. Sauerwein Andrew P. Waters Chris J. Janse 《PLoS pathogens》2010,6(4)
The process of fertilization is critically dependent on the mutual recognition of gametes and in Plasmodium, the male gamete surface protein P48/45 is vital to this process. This protein belongs to a family of 10 structurally related proteins, the so called 6-cys family. To identify the role of additional members of this family in Plasmodium fertilisation, we performed genetic and functional analysis on the five members of the 6-cys family that are transcribed during the gametocyte stage of P. berghei. This analysis revealed that in addition to P48/45, two members (P230 and P47) also play an essential role in the process of parasite fertilization. Mating studies between parasites lacking P230, P48/45 or P47 demonstrate that P230, like P48/45, is a male fertility factor, consistent with the previous demonstration of a protein complex containing both P48/45 and P230. In contrast, disruption of P47 results in a strong reduction of female fertility, while males remain unaffected. Further analysis revealed that gametes of mutants lacking expression of p48/45 or p230 or p47 are unable to either recognise or attach to each other. Disruption of the paralog of p230, p230p, also specifically expressed in gametocytes, had no observable effect on fertilization. These results indicate that the P. berghei 6-cys family contains a number of proteins that are either male or female specific ligands that play an important role in gamete recognition and/or attachment. The implications of low levels of fertilisation that exist even in the absence of these proteins, indicating alternative pathways of fertilisation, as well as positive selection acting on these proteins, are discussed in the context of targeting these proteins as transmission blocking vaccine candidates. 相似文献
95.
Hendrik Schubert Hans C.P. Matthijs Luuc R. Mur Ulrich Schiewer 《FEMS microbiology ecology》1995,18(3):237-245
Abstract: The influence of intermittent high-light dosage on Synechocystis sp. PCC 6803 with respect to oxygen evolution capacity, fluorescence yield and carotenoid pigment pattern was investigated, using high-light- and low-light-adapted cultures. The results showed that this cyanobacterium was able to survive high light stress for a full day if this stress was applied on and off with intermittently presented recovery periods in darkness. Enhanced respiratory activity in the high-light adapted cells was detected and this may be an important factor in preventing photodamage under high light stress. Cyanobacterial photosynthetic and respiratory electron transfer pathways are both present within the same membrane, and share common electron carriers. The role of respiratory activity in preventing overexcitation of photosystem 2 is discussed with regard to cyanobacterial ecology. 相似文献
96.
Velamakanni S Wei SL Janvilisri T van Veen HW 《Journal of bioenergetics and biomembranes》2007,39(5-6):465-471
The ATP-binding cassette (ABC) transporter superfamily is one of the largest protein families with representatives in all
kingdoms of life. Members of this superfamily are involved in a wide variety of transport processes with substrates ranging
from small ions to relatively large polypeptides and polysaccharides. The G subfamily of ABC transporters consists of half-transporters,
which oligomerise to form the functional transporter. While ABCG1, ABCG4 and ABCG5/8 are involved in the ATP-dependent translocation
of steroids and, possibly, other lipids, ABCG2 (also termed the breast cancer resistance protein) has been identified as a
multidrug transporter that confers resistance on tumor cells. Evidence will be summarized suggesting that ABCG2 can also mediate
the binding/transport of non-drug substrates, including free and conjugated steroids. The characterization of the substrate
specificities of ABCG proteins at a molecular level might provide further clues about their potential physiological role(s),
and create new opportunities for the modulation of their activities in relation to human disease. 相似文献
97.
No evidence for substantial aerobic methane emission by terrestrial plants: a 13C-labelling approach 总被引:2,自引:0,他引:2
Dueck TA de Visser R Poorter H Persijn S Gorissen A de Visser W Schapendonk A Verhagen J Snel J Harren FJ Ngai AK Verstappen F Bouwmeester H Voesenek LA van der Werf A 《The New phytologist》2007,175(1):29-35
* The results of a single publication stating that terrestrial plants emit methane has sparked a discussion in several scientific journals, but an independent test has not yet been performed. * Here it is shown, with the use of the stable isotope (13)C and a laser-based measuring technique, that there is no evidence for substantial aerobic methane emission by terrestrial plants, maximally 0.3% (0.4 ng g(-1) h(-1)) of the previously published values. * Data presented here indicate that the contribution of terrestrial plants to global methane emission is very small at best. * Therefore, a revision of carbon sequestration accounting practices based on the earlier reported contribution of methane from terrestrial vegetation is redundant. 相似文献
98.
Ethylene insensitivity results in down-regulation of rubisco expression and photosynthetic capacity in tobacco 总被引:2,自引:0,他引:2 下载免费PDF全文
Little is known about the effect of hormones on the photosynthetic process. Therefore, we studied Rubisco content and expression along with gas exchange parameters in transgenic tobacco (Nicotiana tabacum) plants that are not able to sense ethylene. We also tested for a possible interaction between ethylene insensitivity, abscisic acid (ABA), and sugar feedback on photosynthesis. We measured Rubisco content in seedlings grown in agar with or without added sugar and fluridone, and Rubisco expression in hydroponically grown vegetative plants grown at low and high CO(2). Furthermore, we analyzed gas exchange and the photosynthetic machinery of transformants and wild-type plants grown under standard conditions. In the presence of exogenous glucose (Glc), agar-grown seedlings of the ethylene-insensitive genotype had lower amounts of Rubisco per unit leaf area than the wild type. No differences in Rubisco content were found between ethylene-insensitive and wild-type seedlings treated with fluridone, suggesting that inhibition of ABA production nullified the effect of Glc application. When larger, vegetative plants were grown at different atmospheric CO(2) concentrations, a negative correlation was found between Glc concentration in the leaves and Rubisco gene expression, with stronger repression by high Glc concentrations in ethylene-insensitive plants. Ethylene insensitivity resulted in plants with comparable fractions of nitrogen invested in light harvesting, but lower amounts in electron transport and Rubisco. Consequently, photosynthetic capacity of the insensitive genotype was clearly lower compared with the wild type. We conclude that the inability to perceive ethylene results in increased sensitivity to Glc, which may be mediated by a higher ABA concentration. This increased sensitivity to endogenous Glc has negative consequences for Rubisco content and photosynthetic capacity of these plants. 相似文献
99.