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The reaction parameters and the stereospecificity of the enzymatic cleavage of D,L-5-indolylmethylhydantoin in producing L-tryptophan with resting cells of Arthrobacter sp. DSM 3747 were studied. When intact cells were tested, the optimal pH was between 8.5 and 9.0 and the optimal temperature was 50 degrees C. Both, L-N-carbamoylase and hydantoinase could be stabilized over 24 h at 30 and 40 degrees C by the addition of D,L-5-indolylmethylhydantoin. Furthermore, the hydantoinase was stable over 24 h at 50 degrees C by the addition of 0.5 mM Mn2+ ions. The treatment with sodium desoxycholate turned out to be successful in overcoming the poor availability of D,L-5-indolylmethylhydantoin for the cells. The optimal temperature with permeabilized cells decreased to 30 degrees C and therefore ensured a good enzyme stability. While the L-N-carbamoylase proved to be absolutely L-specific, the hydantoinase led to a mixture of enantiomers of N-carbamoyltryptophan. The produced D-N-carbamoyl-tryptophan caused an inhibition of the L-N-carbamoylase. The transformation yield from D,L-5-indolylmethylhydantoin always reached 100%. 相似文献
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The QRxGRxGRxxxG motif of the vaccinia virus DExH box RNA helicase NPH-II is required for ATP hydrolysis and RNA unwinding but not for RNA binding. 总被引:13,自引:3,他引:10 下载免费PDF全文
Vaccinia virus NPH-II is an essential nucleic acid-dependent nucleoside triphosphate that catalyzes unidirectional unwinding of duplex RNA containing a 3' tail. NPH-II is the prototypal RNA helicase of the DExH box protein family, which is defined by several shared sequence motifs. The contribution of the conserved QRKGRVGRVNPG region to enzyme activity was assessed by alanine-scanning mutagenesis. Ten mutated versions of NPH-II were expressed in vaccinia virus-infected BSC-40 cells and purified by nickel affinity chromatography and glycerol gradient sedimentation. The mutated proteins were characterized with respect to RNA helicase, nucleic acid-dependent ATPase, and RNA binding functions. Individual alanine substitutions at invariant residues Q-491, G-494, R-495, G-497, R-498, and G-502 caused severe defects in RNA unwinding that correlated with reduced rates of ATP hydrolysis. None of these mutations affected the binding of NPH-II to single-strand RNA or to the tailed duplex RNA used as a helicase substrate. Mutation of the strictly conserved position R-492 inhibited ATPase and helicase activities and also caused a modest decrement in RNA binding. Alanine mutations at the nonconserved position N-500 and the weakly conserved residue P-501 had no apparent effect on any activity associated with NPH-II, whereas a mutation at the weakly conserved position K-493 reduced helicase to one-third and ATPase to two-thirds of the activity of wild-type required for ATP hydrolysis and RNA unwinding but not for RNA binding. Because mutations in the HRxGRxxR motif of the prototypal DEAD box RNA helicase eIF-4A abolish or severely inhibit RNA binding, we surmise that the contribution of conserved helicase motifs to overall protein function is context dependent. 相似文献
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Beta-glucan synthesis in Bradyrhizobium japonicum: characterization of a new locus (ndvC) influencing beta-(1-->6) linkages. 下载免费PDF全文
Bradyrhizobium japonicum synthesizes periplasmic cyclic beta-(1-->3),beta-(1-->6)-D-glucans during growth in hypoosmotic environments, and evidence is growing that these molecules may have a specific function during plant-microbe interactions in addition to osmoregulation. Site-directed Tn5 mutagenesis of the DNA region upstream of ndvB resulted in identification of a new gene (ndvC) involved in beta-(1--> 3), beta-(1-->6)-glucan synthesis and in nodule development. The predicted translation product was a polypeptide (ca. 62 kDa) with several transmembrane domains. It contained a sequence characteristic of a conserved nucleoside-sugar-binding motif found in many bacterial enzymes and had 51% similarity with a beta-glucanosyltransferase from Candida albicans. B. japonicum carrying a Tn5 insertion in ndvC resulted in synthesis of altered cyclic beta-glucans composed almost entirely of beta-(1--> 3)-glycosyl linkages. The mutant strain was only slightly sensitive to hypoosmotic growth conditions compared with the ndvB mutant, but it was severely impaired in symbiotic interactions with soybean (Glycine max). Nodulation was delayed by 8 to 10 days, and many small nodule-like structures apparently devoid of viable bacteria were formed. This finding suggests that the structure of the beta-glucan molecule is important for a successful symbiotic interaction, and beta-glucans may have a specific function in addition to their role in hypoosmotic adaptation. 相似文献
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Conserved sequence motifs in the unorthodox BvgS two-component sensor protein ofBordetella pertussis
The unorthodox two-component sensor protein BvgS ofBordetella pertussis contains several interesting sequence motifs of unknown functional relevance, such as a histidine motif in its output domain, which is conserved among several unorthodox sensor proteins, a putative nucleotide binding site [Walker box type A] in its linker region, and a region in its periplasmic domain with significant homology to the TonB protein ofEscherichia coli. We investigated potential functions of these sequences by constructingB. pertussis strains that express mutant BvgS derivatives. The His1172 residue in the output domain was exchanged for Gln, and the Walker motif was mutated either by the replacement of Lys625 by Arg, or of Gly624 by Val and Lys625 by Leu. To analyse the TonB motif, the periplasmic domain of BvgS was replaced with the corresponding domain of EvgS, anE. coli sensor that is highly homologous to BvgS but lacks the similarity with TonB. All mutations except the conservative Lys/Arg exchange in the Walker box caused the inactivation of BvgS, indicating the functional importance of the conserved motifs. The activity of the mutant proteins could be restored by complementation in trans with various separately expressed, truncated parts of BvgS. Mutations in the BvgS receiver domain could be complemented not only by a construct expressing the wild-type receiver and output domains, but also by the derivative containing the His-Gln exchange. Therefore, the histidine motif, although important for BvgS function, is not essential for complementation of BvgS mutants. The mutations in the Walker box and in the periplasmic domain could be complemented by a truncated BvgS derivative lacking the receiver and output domains. The characterization of a spontaneous revertant of the strain expressing the originally inactive EvgS/BvgS hybrid protein revealed the presence of a mutation in the BvgS linker region, conferring constitutive activity on the protein. As TonB energizes transport processes across the outer membrane ofE. coli, the strain expressing the constitutive EvgS/BvgS hybrid protein lacking the TonB motif was used in preliminary investigations of a possible direct involvement of BvgS in transport processes. 相似文献
57.
Gross MR 《Trends in ecology & evolution》1996,11(2):92-98
Not all members of a sex behave in the same way. Frequency- and statusdependent selection have given rise to many alternative reproductive phenotypes within the sexes. The evolution and proximate control of these alternatives are only beginning to be understood. Although game theory has provided a theoretical framework, the concept of the mixed strategy has not been realized in nature, and alternative strategies are very rare. Recent findings suggest that almost all alternative reproductive phenotypes within the sexes are due to alternative tactics within a conditional strategy, and, as such, while the average fitnesses of the alternative phenotypes are unequal, the strategy is favoured in evolution. Proximate mechanisms that underlie alternative phenotypes may have many similarities with those operating between the sexes. 相似文献
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Thomas Schnyder Peter Tittmann Hanspeter Winkler Heinz Gross Theo Wallimann 《Journal of structural biology》1995,114(3)
Octamers of mitochondrial creatine kinase (Mi-CK) wore modified with the thiol-specific reagents N-ethylmaleimide or the gold-coupled derivative, maleidoyl undecagold. The kinetics of inhibition of the Mi-CK catalysis was shown to be comparable for both reagents, suggesting that the large gold cluster complex is accessible to the reactive cysteines. SDS-PAGE analysis revealed that two of eight cysteines per Mi-CK monomer were labeled with maleidoyl undecagold with a similar affinity for the functional maleimide group. Gel exclusion chromatography of labeled molecules showed that the octameric structure of Mi-CK was preserved after thiol modification. Freeze-dried gold-labeled octamers visualized by electron microscopy under cryoconditions were enhanced in contrast and showed a well-preserved fourfold symmetry of the end-on view, Image analysis of gold-labeled Mi-CK exhibited an averaged end-on view with four strong contrast signals located at the periphery of the notamer, whereas the center of the molecule remained electron translucent. We conclude that the two cysteine residues per monomer labeled with maleidoyl undecagold are located at the octamer's perimeter and we discuss the possible role of these reactive cysteines in enzyme catalysis. 相似文献