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1.
The three dimensional model of cold-adapted Alaskan psychrotroph Pseudomonas species (Strain B11-1) lipase has been constructed by homology modeling based on the crystal structure of acetyl esterase from Rhodococcus species and refined by molecular dynamics methods. Our model locates the substrate-binding cavity and further suggests that Ser-155, Asp-250, and His-280 are the members of the catalytic triad. Substrate specificity of the modeled lipase has been examined by docking experiments, which indicates that the ester of C(6) fatty acid has the highest affinity for the enzyme. Our model also identifies the oxyanion hole that plays an important role in the stabilization of the tetrahedral intermediate during catalysis. Comparison of this cold-adapted lipase with the crystal structure of a thermophilic Bacillus stearothermophilus P1 lipase supported the assumption that cold-adapted enzymes have a more flexible three-dimensional structure than their thermophilic counterparts. The conformational flexibility of this modeled cold-adapted lipase at low temperature probably originates from a combination of factors compared to its thermophilic counterpart, i.e., lower number of salt bridges and cation-pi interactions, increase in the non-polar surface area exposed to solvent. Our study may help in understanding the structural features of a cold-adapted lipase and can further be used in engineering lipase that can function at or near extreme temperatures with considerable biotechnological potential. 相似文献
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低温微生物适应低温的分子机制 总被引:7,自引:0,他引:7
根据Morita和Russel的定义 ,低温微生物可分为两类 :一类是其最高生长温度低于2 0℃的微生物称嗜冷菌 (psychrophiles) ,另一类是指在 0~ 40℃可以生长的微生物称耐冷菌 (psychrotrophs)。这些微生物体内的温度与环境温度很接近。尽管低温对生化反应有着强烈的负效应 ,但低温微生物在低温下却能成功地生长与繁殖 ,它们因此产生了各种各样的适应机制 ,主要是细胞膜、蛋白质、酶分子水平上发生了精细的组成和结构的变化[1] ,因此弥补了低温对生长的有害影响。催化生物体内所有生化反应的酶是生物体适… 相似文献
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Ping-Yi Li Peng Ji Chun-Yang Li Yi Zhang Guang-Long Wang Xi-Ying Zhang Bin-Bin Xie Qi-Long Qin Xiu-Lan Chen Bai-Cheng Zhou Yu-Zhong Zhang 《The Journal of biological chemistry》2014,289(27):19031-19041
Hormone-sensitive lipases (HSLs) are widely distributed in microorganisms, plants, and animals. Microbial HSLs are classified into two subfamilies, an unnamed new subfamily and the GDSAG motif subfamily. Due to the lack of structural information, the detailed catalytic mechanism of the new subfamily is not yet clarified. Based on sequence analysis, we propose to name the new subfamily as the GTSAG motif subfamily. We identified a novel HSL esterase E25, a member of the GTSAG motif subfamily, by functional metagenomic screening, and resolved its structure at 2.05 Å. E25 is mesophilic (optimum temperature at 50 °C), salt-tolerant, slightly alkaline (optimum pH at 8.5) for its activity, and capable of hydrolyzing short chain monoesters (C2–C10). E25 tends to form dimers both in the crystal and in solution. An E25 monomer contains an N-terminal CAP domain, and a classical α/β hydrolase-fold domain. Residues Ser186, Asp282, and His312 comprise the catalytic triad. Structural and mutational analyses indicated that E25 adopts a dimerization pattern distinct from other HSLs. E25 dimer is mainly stabilized by an N-terminal loop intersection from the CAP domains and hydrogen bonds and salt bridges involving seven highly conserved hydrophilic residues from the catalytic domains. Further analysis indicated that E25 also has some catalytic profiles different from other HSLs. Dimerization is essential for E25 to exert its catalytic activity by keeping the accurate orientation of the catalytic Asp282 within the catalytic triad. Our results reveal the structural basis for dimerization and catalysis of an esterase from the GTSAG motif subfamily of the HSL family. 相似文献
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Colette Breuil D. B. Shindler J. S. Sijher D. J. Kushner 《Journal of bacteriology》1978,133(2):601-606
Acinetobacter lwoffi strain O16, a facultative psychrophile, can grow on crude oil, hexadecane, octadecane, and most alkanes when tested at 20 but not at 30°C. Growth occurred on a few alkanes at 30°C but after a longer lag than at 20°C. Cells grown on alkanes as sole carbon sources had high levels of cell-bound lipase. In contrast, previous work has shown that those grown on complex medium produced cell-free lipase and those grown on defined medium without alkanes produced little or no lipase. Low concentrations of the detergent Triton X-100 caused the liberation of most of the lipase activity of alkane-grown cells and increased total lipase activity. When ethanol and hexadecane were both present in a mineral medium, diauxic growth occurred; until the ethanol was completely used up, hexadecane was not utilized, and the lipase activity was very low. When growth on hexadecane began, lipase activity increased, reaching a level 50- to 100-fold higher than that of cells growing on ethanol. A similar pattern of lipase formation and hexadecane utilization was observed with Pseudomonas aeruginosa. Whenever A. lwoffi and other bacteria degraded alkanes they exhibited substantial lipase activity. Not all bacteria that produced lipase, however, could attack alkanes. Bacteria that could not produce lipase did not attack alkanes. The results suggest that a correlation may exist between lipase formation and alkane utilization. 相似文献
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Srinivasan Rengachari Philipp Aschauer Matthias Schittmayer Nicole Mayer Karl Gruber Rolf Breinbauer Ruth Birner-Gruenberger Ingrid Dreveny Monika Oberer 《The Journal of biological chemistry》2013,288(43):31093-31104
Monoacylglycerol lipases (MGLs) play an important role in lipid catabolism across all kingdoms of life by catalyzing the release of free fatty acids from monoacylglycerols. The three-dimensional structures of human and a bacterial MGL were determined only recently as the first members of this lipase family. In addition to the α/β-hydrolase core, they showed unexpected structural similarities even in the cap region. Nevertheless, the structural basis for substrate binding and conformational changes of MGLs is poorly understood. Here, we present a comprehensive study of five crystal structures of MGL from Bacillus sp. H257 in its free form and in complex with different substrate analogs and the natural substrate 1-lauroylglycerol. The occurrence of different conformations reveals a high degree of conformational plasticity of the cap region. We identify a specific residue, Ile-145, that might act as a gatekeeper restricting access to the binding site. Site-directed mutagenesis of Ile-145 leads to significantly reduced hydrolase activity. Bacterial MGLs in complex with 1-lauroylglycerol, myristoyl, palmitoyl, and stearoyl substrate analogs enable identification of the binding sites for the alkyl chain and the glycerol moiety of the natural ligand. They also provide snapshots of the hydrolytic reaction of a bacterial MGL at different stages. The alkyl chains are buried in a hydrophobic tunnel in an extended conformation. Binding of the glycerol moiety is mediated via Glu-156 and water molecules. Analysis of the structural features responsible for cap plasticity and the binding modes of the ligands suggests conservation of these features also in human MGL. 相似文献
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T. Bertrand F. Augé A. Rak V. Mikol N. Michot C. Hoornaert 《Journal of molecular biology》2010,396(3):663-673
Monoglyceride lipase (MGL) is a serine hydrolase that hydrolyses 2-arachidonoylglycerol (2-AG) into arachidonic acid and glycerol. 2-AG is an endogenous ligand of cannabinoid receptors, involved in various physiological processes in the brain. We present here the first crystal structure of human MGL in its apo form and in complex with the covalent inhibitor SAR629. MGL shares the classic fold of the α/β hydrolase family but depicts an unusually large hydrophobic occluded tunnel with a highly flexible lid at its entry and the catalytic triad buried at its end. Structures reveal the configuration of the catalytic triad and the shape and nature of the binding site of 2-AG. The bound structure of SAR629 highlights the key interactions for productive binding with MGL. The shape of the tunnel suggests a high druggability of the protein and provides an attractive template for drug discovery. 相似文献
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Leandro A. Sánchez Fiorella F. Gómez Osvaldo D. Delgado 《Extremophiles : life under extreme conditions》2009,13(1):111-120
Thirty out of 8,000 different colony morphotypes isolated from soil samples of Isla de los Estados were selected based on
their ability to produce antimicrobials. The significant influence of culture media and incubation temperature on antimicrobial
production was proved, being LB medium and 8°C the conditions of choice. Most of the psychrotolerant isolates were phylogenetically
related to Serratia proteamaculans (96.4–97.9%) while the psychrophilic isolated 8H1 was closely related to Pseudomonas sp. (90–94% similarity). Produced antimicrobials showed a promising wide spectrum of activity both against gram-positive
and gram-negative pathogenic bacteria. They were suspected to be microcin-like compounds (Mw <2,000 Da) and showed a marked
tolerance to heat (1 h in boiling water bath) and pH-treatments (1–12). Antimicrobial compounds also showed to partially keep
their activity even after overnight freezing at −20 and −80°C and displayed a negative net charge at pH 8.0, a common feature
of class II microcins. 相似文献
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In this study, we assessed various leaf structural and chemical features as possible predictors of the size of the phyllosphere
bacterial population in the Mediterranean environment. We examined eight perennial species, naturally occurring and coexisting
in the same area, in Halkidiki (northern Greece). They are Arbutus unedo, Quercus coccifera, Pistacia lentiscus, and Myrtus communis (evergreen sclerophyllous species), Lavandula stoechas and Cistus incanus (drought semideciduous species), and Calamintha nepeta and Melissa officinalis (nonwoody perennial species). M. communis, L. stoechas, C. nepeta, and M. officinalis produce essential oil in substantial quantities. We sampled summer leaves from these species and (1) estimated the size of
the bacterial population of their phyllosphere, (2) estimated the concentration of different leaf constituents, and (3) studied
leaf morphological and anatomical features and expressed them in a quantitative way. The aromatic plants are on average more
highly colonized than the other species, whereas the nonwoody perennials are more highly colonized than the woody species.
The population size of epiphytic bacteria is positively correlated with glandular and nonglandular trichome densities, and
with water and phosphorus contents; it is negatively correlated with total phenolics content and the thickness of the leaf,
of the mesophyll, and of the abaxial epidermis. No correlation was found with the density of stomata, the nitrogen, and the
soluble sugar contents. By regression tree analysis, we found that the leaf-microbe system can be effectively described by
three leaf attributes with leaf water content being the primary explanatory attribute. Leaves with water content >73% are
the most highly colonized. For leaves with water content <73%, the phosphorus content, with a critical value of 1.34 mg g−1 d.w., is the next explanatory leaf attribute, followed by the thickness of the adaxial epidermis. Leaves higher in phosphorus
(>1.34 mg g−1 d.w.) are more colonized, and leaves with the adaxial epidermis thicker than 20.77 μm are the least colonized. Although these
critical attributes and values hold true only within the Mediterranean ecosystem studied and the range of observations taken,
they are important because they provide a hypothesis to be tested in other Mediterranean ecosystems and other biomes. Such
comparative studies may give insight as to the general properties governing the leaf-microbe system. 相似文献
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Rabeb Dhouib Fran?oise Laval Frédéric Carrière Mamadou Daffé Stéphane Canaan 《Journal of bacteriology》2010,192(18):4776-4785
MSMEG_0220 from Mycobacterium smegmatis, the ortholog of the Rv0183 gene from M. tuberculosis, recently identified and characterized as encoding a monoacylglycerol lipase, was cloned and expressed in Escherichia coli. The recombinant protein (rMSMEG_0220), which exhibits 68% amino acid sequence identity with Rv0183, showed the same substrate specificity and similar patterns of pH-dependent activity and stability as the M. tuberculosis enzyme. rMSMEG_0220 was found to hydrolyze long-chain monoacylglycerol with a specific activity of 143 ± 6 U mg−1. Like Rv0183 in M. tuberculosis, MSMEG_0220 was found to be located in the cell wall. To assess the in vivo role of the homologous proteins, an MSMEG_0220 disrupted mutant of M. smegmatis (MsΔ0220) was produced. An intriguing change in the colony morphology and in the cell interaction, which were partly restored in the complemented mutant containing either an active (ComMsΔ0220) or an inactive (ComMsΔ0220S111A) enzyme, was observed. Growth studies performed in media supplemented with monoolein showed that the ability of both MsΔ0220 and ComMsΔ0220S111A to grow in the presence of this lipid was impaired. Moreover, studies of the antimicrobial susceptibility of the MsΔ0220 strain showed that this mutant is more sensitive to rifampin and more resistant to isoniazid than the wild-type strain, pointing to a critical structural role of this enzyme in mycobacterial physiology, in addition to its function in the hydrolysis of exogenous lipids.Tuberculosis, which is caused by Mycobacterium tuberculosis, is a major public health issue worldwide. Because of the emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains and the high incidence of HIV and tuberculosis coinfection (16), it is becoming increasingly difficult to combat the spread of this disease, and the global health burden of tuberculosis is extremely heavy. The reasons for the persistence of the tubercle bacillus include not only its ability to enter into a state of dormancy in its host for decades, evading the immune system by forming structures called granulomas (17), but also its unique and complex cell wall composed of specific lipids (8). These characteristics are thought to be good focus points for drug development. In granulomas, during the nonreplicative stage, the bacteria have been found to accumulate lipids in the form of intracellular lipid inclusion bodies (LIBs) (13). These lipids are composed mainly of triacylglycerols (TAG) (9, 13) and may originate from the lipolysis of host lipids and/or fatty acid uptake. In fact, M. tuberculosis in the granuloma center can even accumulate lipids originating from the degradation of immune cells (20). In addition, it has been reported that M. tuberculosis internalized by foamy macrophages accumulated LIBs when it joined cell lipid droplets composed of neutral lipids (32). Lipid storage may provide the bacillus with energy via the β-oxidation pathway followed by the glyoxylate cycle, during the chronic phase and the reactivation step (3, 17). These lipids may also supply precursors for the synthesis of bacterial cell membrane lipids, which play a key role in the pathogenicity of M. tuberculosis (4, 23). To investigate the molecular basis of the virulence and pathogenicity of M. tuberculosis, it was therefore proposed to study the lipid metabolism and cell wall remodeling processes in this bacterium.The enzymes involved in the lipid degradation processes induced by this bacterium have attracted considerable attention during the last few years. Based on the complete M. tuberculosis H37Rv genome sequence (6), several open reading frames (ORFs) encoding proteins potentially involved in the lipid metabolism of this strain have been identified, among which are the two lipases from M. tuberculosis that have been purified and characterized so far. Deb et al. identified an enzyme, Rv3097c (LipY), belonging to the hormone-sensitive lipase family, which is able to hydrolyze long-chain TAG (10). A study of LIB mobilization in a lipY-deficient mutant has shown that LipY was involved in TAG hydrolysis under nutriment-deprived conditions (10). LipY may therefore be involved in the degradation of TAG stored during the dormant stage and the subsequent reactivation of the pathogen. In addition, electron microscopy immunolabeling studies of LipY clearly showed that the enzyme had a cell surface localization, thus in direct contact with the host immune system (28). The last identified lipase to date is a monoacylglycerol lipase annotated Rv0183 (7). Like LipY, Rv0183 is located in the cell wall, but its exact physiological function has not yet been elucidated. One hypothesis could be that, like some mammalian cells (e.g., adipocytes), M. tuberculosis expresses several lipolytic enzymes sequentially involved in the lipolysis of TAG (37). The Rv0183 enzyme is conserved in M. bovis (Mb0189) and M. leprae (ML2603), as well as in M. smegmatis (MSMEG_0220), a nonpathogenic mycobacterium which provides a useful model organism and a surrogate host for molecular analysis of M. tuberculosis (19). In order to decipher the cellular role of Rv0183 in M. tuberculosis H37Rv and its contribution to the lipid metabolism of this bacterium, biochemical studies were performed on the homologue MSMEG_0220. For this purpose, the MSMEG_0220 gene from M. smegmatis, encoding a protein showing 68% amino acid sequence identity with Rv0183, was cloned, and the recombinant MSMEG_0220 enzyme (rMSMEG_0220) was produced in Escherichia coli, purified, and biochemically characterized. An M. smegmatis mutant with an MSMEG_0220 disrupted gene was produced to investigate the physiological role of MSMEG_0220. 相似文献
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Bacterial Microcompartments (BMCs) are proteinaceous organelles that encapsulate critical segments of autotrophic and heterotrophic metabolic pathways; they are functionally diverse and are found across 23 different phyla. The majority of catabolic BMCs (metabolosomes) compartmentalize a common core of enzymes to metabolize compounds via a toxic and/or volatile aldehyde intermediate. The core enzyme phosphotransacylase (PTAC) recycles Coenzyme A and generates an acyl phosphate that can serve as an energy source. The PTAC predominantly associated with metabolosomes (PduL) has no sequence homology to the PTAC ubiquitous among fermentative bacteria (Pta). Here, we report two high-resolution PduL crystal structures with bound substrates. The PduL fold is unrelated to that of Pta; it contains a dimetal active site involved in a catalytic mechanism distinct from that of the housekeeping PTAC. Accordingly, PduL and Pta exemplify functional, but not structural, convergent evolution. The PduL structure, in the context of the catalytic core, completes our understanding of the structural basis of cofactor recycling in the metabolosome lumen. 相似文献
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Novel Cold-Adapted Alkaline Lipase from an Intertidal Flat Metagenome and Proposal for a New Family of Bacterial Lipases 总被引:1,自引:0,他引:1
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Eun-Young Kim Ki-Hoon Oh Mi-Hwa Lee Chul-Hyung Kang Tae-Kwang Oh Jung-Hoon Yoon 《Applied microbiology》2009,75(1):257-260
A new lipase, LipEH166, isolated from an intertidal flat metagenome, showed no amino acid similarity to any known lipolytic enzyme except in the consensus region. This suggested that LipEH166 and its homologues belong to a new family of lipolytic enzymes. Partial characterization indicated that LipEH166 is a novel cold-adapted alkaline lipase. 相似文献
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Anna I. Podgornaia Patricia Casino Alberto Marina Michael T. Laub 《Structure (London, England : 1993)》2013,21(9):1636-1647
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18.
Juntaek Oh Eunhye Goo Ingyu Hwang Sangkee Rhee 《The Journal of biological chemistry》2014,289(16):11465-11475
The Burkholderia species utilize acetyl-CoA and oxaloacetate, substrates for citrate synthase in the TCA cycle, to produce oxalic acid in response to bacterial cell to cell communication, called quorum sensing. Quorum sensing-mediated oxalogenesis via a sequential reaction by ObcA and ObcB counteracts the population-collapsing alkaline pH of the stationary growth phase. Thus, the oxalic acid produced plays an essential role as an excreted public good for survival of the group. Here, we report structural and functional analyses of ObcA, revealing mechanistic features distinct from those of citrate synthase. ObcA exhibits a unique fold, in which a (β/α)8-barrel fold is located in the C-domain with the N-domain inserted into a loop following α1 in the barrel fold. Structural analyses of the complexes with oxaloacetate and with a bisubstrate adduct indicate that each of the oxaloacetate and acetyl-CoA substrates is bound to an independent site near the metal coordination shell in the barrel fold. In catalysis, oxaloacetate serves as a nucleophile by forming an enolate intermediate mediated by Tyr322 as a general base, which then attacks the thioester carbonyl carbon of acetyl-CoA to yield a tetrahedral adduct between the two substrates. Therefore, ObcA catalyzes its reaction by combining the enolase and acetyltransferase superfamilies, but the presence of the metal coordination shell and the absence of general acid(s) produces an unusual tetrahedral CoA adduct as a stable product. These results provide the structural basis for understanding the first step in oxalogenesis and constitute an example of the functional diversity of an enzyme for survival and adaptation in the environment. 相似文献
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Marsh L 《Journal of molecular evolution》2006,62(5):575-587
The globin family of proteins has a characteristic structural pattern of helix interactions that nonetheless exhibits some
variation. A simplified model for globin structural evolution was developed in which protein shape evolved by random change
of contacts between helices. A conserved globin domain of 15 bacterial proteins representing four structural families was
studied. Using a parsimony approach ancestral structural states could be reconstructed. The distribution of number of contact
changes per site for a fixed topology tree fit a gamma distribution. Homoplasy was high, with multiple changes per site and
no support for an invariant class of residue-residue contacts. Contacts changed more slowly than sequence. A phylogenetic
reconstruction using a distance measure based on the proportion of shared contacts was generally consistent with a sequence-based
phylogeny but not highly resolved. Contact pattern convergence between members of different globin family proteins could not
be detected. Simulation studies indicated the convergence test was sensitive enough to have detected convergence involving
only 10% of the contacts, suggesting a limit on the extent of selection for a specific contact pattern. Contact site methods
may provide additional approaches to study the relationship between protein structure and sequence evolution.
[Reviewing Editior: Dr. Lauren Ancel Meyers] 相似文献