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991.
Amino acid replacement in a mutant lipoprotein of the Escherichia coli outer membrane. 总被引:3,自引:0,他引:3
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S Inouye N Lee M Inouye H C Wu H Suzuki Y Nishimura H Iketani Y Hirota 《Journal of bacteriology》1977,132(1):308-313
The primary structure of a mutant lipoprotein of the outer membrane of Escherichia coli was investigated. This mutant was previously described as a mutant that forms a dimer of the lipoprotein by an S-S bridge (H. Suzuki et al., J. Bacteriol. 127:1494-1501, 1976). The amino acid analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of the mutant lipoprotein revealed that the mutant lipoprotein had an extra cysteine residue, with concomitant loss of an arginine residue. From the analysis of tryptic peptides, it was found that the arginine residue at position 57 was replaced with a cysteine residue. The amino terminal structure of the mutant lipoprotein was found to be glycerylcysteine, as in the case of the wild-type lipoprotein. The present results show that the mutation that was previously determined to map at 36.5 min on the E. coli chromosome occurred in the structure gene (lpp) for the lipoprotein. This was further confirmed by the fact that a merodiploid carrying both lpp+ and lpp produces not only the wild-type lipoprotein but also the mutant lipoprotein. 相似文献
992.
993.
In Escherichia coli, the cold shock response is exerted upon a temperature change from 37°C to 15°C and is characterized by induction of several cold shock proteins, including polynucleotide phosphorylase (PNPase), during acclimation phase. In E. coli, PNPase is essential for growth at low temperatures; however, its exact role in this essential function has not been fully elucidated. PNPase is a 3′-to-5′ exoribonuclease and promotes the processive degradation of RNA. Our screening of an E. coli genomic library for an in vivo counterpart of PNPase that can compensate for its absence at low temperature revealed only one protein, another 3′-to-5′ exonuclease, RNase II. Here we show that the RNase PH domains 1 and 2 of PNPase are important for its cold shock function, suggesting that the RNase activity of PNPase is critical for its essential function at low temperature. We also show that its polymerization activity is dispensable in its cold shock function. Interestingly, the third 3′-to-5′ processing exoribonuclease, RNase R of E. coli, which is cold inducible, cannot complement the cold shock function of PNPase. We further show that this difference is due to the different targets of these enzymes and stabilization of some of the PNPase-sensitive mRNAs, like fis, in the Δpnp cells has consequences, such as accumulation of ribosomal subunits in the Δpnp cells, which may play a role in the cold sensitivity of this strain. 相似文献
994.
An ultimate goal for any protein production system is to express only the protein of interest without producing other cellular proteins. To date, there are only two established methods that will allow the successful expression of only the protein of interest: the cell-free in vitro protein synthesis system and the in vivo single-protein production (SPP) system. Although single-protein production can be achieved in cell-free systems, it is not easy to completely suppress the production of cellular proteins during the production of a protein of interest in a living cell. However, the finding of a unique sequence-specific mRNA interferase in Escherichia coli led to the development of the SPP system by converting living cells into a bioreactor that produces only a single protein of interest without producing any cellular proteins. This technology not only provides a new high expression system for proteins, but also offers a novel avenue for protein structural studies. 相似文献
995.
996.
A unique GTP-binding protein, Der contains two consecutive GTP-binding domains at the N-terminal region and its homologues are highly conserved in eubacteria but not in archaea and eukaryotes. In the present paper, we demonstrate that Der is one of the essential GTPases in Escherichia coli and that the growth rate correlates with the amount of Der in the cell. Interestingly, both GTP-binding domains are required at low temperature for cell growth, while at high temperature either one of the two domains is dispensable. Result of the sucrose density gradient experiment suggests that Der interacts specifically with 50S ribosomal subunits only in the presence of a GTP analogue, GMPPNP. The depletion of Der accumulates 50S and 30S ribosomal subunits with a concomitant reduction of polysomes and 70S ribosomes. Notably, Der-depleted cells accumulate precursors of both 23S and 16S rRNAs. Moreover, at lower Mg2+ concentration, 50S ribosomal subunits from Der-depleted cells are further dissociated into aberrant 50S ribosomal subunits; however, 30S subunits are stable. It was revealed that the aberrant 50S subunits, 40S subunits, contain less ribosomal proteins with significantly reduced amounts of L9 and L18. These results suggest that Der is a novel 50S ribosome-associated factor involved in the biogenesis and stability of 50S ribosomal subunits. We propose that Der plays a pivotal role in ribosome biogenesis possibly through interaction with rRNA or rRNA/r-protein complex. 相似文献
997.
Masayuki Kusano Kiyoshi Yasukawa Kuniyo Inouye 《Enzyme and microbial technology》2010,46(3-4):320-325
Thermolysin is industrially used for the synthesis of N-carbobenzoxy-l-aspartyl-l-phenylalanine methyl ester (ZDFM), a precursor of an artificial sweetener, aspartame, from N-carbobenzoxy-l-aspartic acid (ZD) and l-phenylalanine methyl ester (FM). We have reported five thermolysin variants [D150A (Asp150 is replaced with Ala), D150E, D150W, I168A, and N227H] with improved activity generated by site-directed mutagenesis of the residues located at the active site [Kusano et al. J Biochem 2009;145:103–13]. In this study, we analyzed the ZDFM synthesis reaction catalyzed by these variants. Steady-state kinetic analysis revealed that in the ZDFM synthesis reaction at pH 7.5, at 25 °C, the molecular activity kcat values of the variants were 1.6–3.8 times higher than that of the wild-type thermolysin (WT), while their Michaelis constant Km values for ZD and FM were almost the same as those of WT. With the initial concentrations of enzyme, ZD, and FM of 0.1 μM, 5 mM, and 5 mM, respectively, the synthesis of ZDFM catalyzed by these variants reached the maximum level at 4 h while that catalyzed by WT did at 12 h. These results suggest that the five thermolysin variants examined are more suitable than WT for use in ZDFM synthesis. 相似文献
998.
999.
Luo X Cerullo J Dawli T Priest C Haddadin Z Kim A Inouye H Suffoletto BP Avila RL Lees JP Sharma D Xie B Costello CE Kirschner DA 《Journal of structural biology》2008,162(1):170-183
P0 glycoprotein is the major structural protein of peripheral nerve myelin where it is thought to modulate inter-membrane adhesion at both the extracellular apposition, which is labile upon changes in pH and ionic strength, and the cytoplasmic apposition, which is resistant to such changes. Most studies on P0 have focused on structure-function correlates in higher vertebrates. Here, we focused on its role in the structure and interactions of frog (Xenopus laevis) myelin, where it exists primarily in a dimeric form. As part of our study, we deduced the full sequence of X. laevis P0 (xP0) from its cDNA. The xP0 sequence was found to be similar to P0 sequences of higher vertebrates, suggesting that a common mechanism of PNS myelin compaction via P0 interaction might have emerged through evolution. As previously reported for mouse PNS myelin, a similar change of extracellular apposition in frog PNS myelin as a function of pH and ionic strength was observed, which can be explained by a conformational change of P0 due to protonation-deprotonation of His52 at P0's putative adhesive interface. On the other hand, the cytoplasmic apposition in frog PNS myelin, like that in the mouse, remained unchanged at different pH and ionic strength. The contribution of hydrophobic interactions to stabilizing the cytoplasmic apposition was tested by incubating sciatic nerves with detergents. Dramatic expansion at the cytoplasmic apposition was observed for both frog and mouse, indicating a common hydrophobic nature at this apposition. Urea also expanded the cytoplasmic apposition of frog myelin likely owing to denaturation of P0. Removal of the fatty acids that attached to the single Cys residue in the cytoplasmic domain of P0 did not change PNS myelin structure of either frog or mouse, suggesting that the P0-attached fatty acyl chain does not play a significant role in PNS myelin compaction and stability. These results help clarify the present understanding of P0's adhesion role and the role of its acylation in compact PNS myelin. 相似文献
1000.
The mRNA interferases, MazF-mt3 and MazF-mt7 from Mycobacterium tuberculosis target unique pentad sequences in single-stranded RNA 总被引:2,自引:0,他引:2
mRNA interferases are sequence-specific endoribonucleases encoded by toxin-antitoxin (TA) systems in bacterial genomes. Previously, we demonstrated that Mycobacterium tuberculosis contains at least seven genes encoding MazF homologues (MazF-mt1 to -mt7) and determined cleavage specificities for MazF-mt1 and MazF-mt6. Here we have developed a new general method for the determination of recognition sequences longer than three bases for mRNA interferases with the use of phage MS2 RNA as a substrate and CspA, an RNA chaperone, which prevents the formation of secondary structures in the RNA substrate. Using this method, we determined that MazF-mt3 cleaves RNA at UU˘CCU or CU˘CCU and MazF-mt7 at U˘CGCU (˘indicates the cleavage site). As pentad sequence recognition is more specific than those of previously characterized mRNA interferases, bioinformatics analysis was carried out to identify M. tuberculosis mRNAs that may be resistant to MazF-mt3 and MazF-mt7 cleavage. The pentad sequence was found to be significantly underrepresented in several genes, including members of the PE and PPE families, large families of proteins that play a role in tuberculosis immunity and pathogenesis. These data suggest that MazF-mt3 and MazF-mt7 or other mRNA interferases that target longer RNA sequences may alter protein expression through differential mRNA degradation, a regulatory mechanism that may allow adaptation to environmental conditions, including those encountered by pathogens such as M. tuberculosis during infection. 相似文献