New Thermophilic and Thermostable Esterase with Sequence Similarity to the Hormone-Sensitive Lipase Family, Cloned from a Metagenomic Library

Rhee, Jin Kyu; Ahn, Dong‐Gyu; Kim, Yeon Gu; Oh, Jisun

doi:10.1128/aem.71.2.817-825.2005

Cited by 214 publications

(152 citation statements)

References 46 publications

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“…Using this approach, several groups isolated novel enzymes from various environments (Ranjan et al, 2005;Rhee et al, 2005;Feng et al, 2007;Hardeman and Sjoling, 2007;Tirawongsaroj et al, 2008), and many of them were found to offer a good potential as new tools for industrial applications, because their biophysical properties arise from improved adaptations to the conditions met in the environment during the sampling process. Although such studies reported the characterization of metagenome-derived enzymes, insights into their physiological aspect are often lacking.…”

Section: Introductionmentioning

confidence: 99%

Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples

et al. 2009

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In this study, the mining of an Antarctic soil sample by functional metagenomics allowed the isolation of a cold-adapted protein (RBcel1) that hydrolyzes only carboxymethyl cellulose. The new enzyme is related to family 5 of the glycosyl hydrolase (GH5) protein from Pseudomonas stutzeri (Pst_2494) and does not possess a carbohydrate-binding domain. The protein was produced and purified to homogeneity. RBcel1 displayed an endoglucanase activity, producing cellobiose and cellotriose, using carboxymethyl cellulose as a substrate. Moreover, the study of pH and the thermal dependence of the hydrolytic activity shows that RBcel1 was active from pH 6 to pH 9 and remained significantly active when temperature decreased (18% of activity at 10 1C). It is interesting that RBcel1 was able to synthetize non-reticulated cellulose using cellobiose as a substrate. Moreover, by a combination of bioinformatics and enzyme analysis, the physiological relevance of the RBcel1 protein and its mesophilic homologous Pst_2494 protein from P. stutzeri, A1501, was established as the key enzymes involved in the production of cellulose by bacteria. In addition, RBcel1 and Pst_2494 are the two primary enzymes belonging to the GH5 family involved in this process.

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Section: Introductionmentioning

confidence: 99%

Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples

et al. 2009

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“…The amino acid sequence of the deduced protein showed only moderate identity (64.8%) to known esterases/ lipases in the database. A gene coding for a thermostable esterase (estE1) has been isolated from mud and water rich sediment of a hot water spring [95]. The enzyme showed high thermal stability and was active between 30° and 95° C. It showed 64% homology to an enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis.…”

Section: Enzyme and Natural Productsmentioning

confidence: 99%

“…The drawback of the sequence-driven approach is overcome here, as both novel and previously described genes can be identifi ed through this method. Some genes that have been detected by virtue of functional screening are oxidoreductases [81], amylases [84], lipases [81], and esterases [95,96]. The identifi cation of clones which express benefi cial natural products or proteins fi nd direct application in the industry and emphasizes on the need of high-throughput screening technologies specifi c for required products.…”

Section: Screening Of Metagenomic Librariesmentioning

confidence: 99%

From bacterial genomics to metagenomics: concept, tools and recent advances

et al. 2008

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In the last 20 years, the applications of genomics tools have completely transformed the fi eld of microbial research. This has primarily happened due to revolution in sequencing technologies that have become available today. This review therefore, fi rst describes the discoveries, upgradation and automation of sequencing techniques in a chronological order, followed by a brief discussion on microbial genomics. Some of the recently sequenced bacterial genomes are described to explain how complete genome data is now being used to derive interesting fi ndings. Apart from the genomics of individual microbes, the study of unculturable microbiota from different environments is increasingly gaining importance. The second section is thus dedicated to the concept of metagenomics describing environmental DNA isolation, metagenomic library construction and screening methods to look for novel and potentially important genes, enzymes and biomolecules. It also deals with the pioneering studies in the area of metagenomics that are offering new insights into the previously unappreciated microbial world.

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“…HSL-like carboxylesterases (EC 3.1.1.1) target ester bonds from short fatty acid chains, and their activity falls dramatically in chains longer than 8 or 10 carbons (56,57). By contrast, lipases (EC 3.1.1.3) hydrolyze ester bonds from fatty acid carbon chains with more than 10 carbons (58,59).…”

Section: Rmsd (å)mentioning

confidence: 99%

Prediction and experimental validation of enzyme substrate specificity in protein structures

Amin

Erdin

Ward³

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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Structural Genomics aims to elucidate protein structures to identify their functions. Unfortunately, the variation of just a few residues can be enough to alter activity or binding specificity and limit the functional resolution of annotations based on sequence and structure; in enzymes, substrates are especially difficult to predict. Here, large-scale controls and direct experiments show that the local similarity of five or six residues selected because they are evolutionarily important and on the protein surface can suffice to identify an enzyme activity and substrate. A motif of five residues predicted that a previously uncharacterized Silicibacter sp. protein was a carboxylesterase for short fatty acyl chains, similar to hormone-sensitive-lipase-like proteins that share less than 20% sequence identity. Assays and directed mutations confirmed this activity and showed that the motif was essential for catalysis and substrate specificity. We conclude that evolutionary and structural information may be combined on a Structural Genomics scale to create motifs of mixed catalytic and noncatalytic residues that identify enzyme activity and substrate specificity. A s the list of known genes grows exponentially, the elucidation of their function remains a major bottleneck and lags far behind the production of sequences (1-5). The best approach remains to search computationally for functionally characterized sequence homologs, ideally with greater than 50% sequence identity (6). Binding specificity, however, is sensitive to subtle amino acid differences, and the transfer of substrate between related enzymes is prone to errors when sequence identity is below 65-80% (7-9). These thresholds vary from case to case: Some orthologs will maintain identical functions down to 25% sequence identify (9), whereas paralogs can take on highly diverse activities (10). Other difficulties that plague annotation transfer between homologs are that individual small molecules may each bind to multiple and distinct molecular pockets (11), that different residues can support similar chemistries (12), and that activity can vary even when catalytic residues are conserved (13-18). To raise annotation accuracy, Structural Genomics (19) made structural information widely available and spurred the development of annotation methods dependent on local chemical and physical environments (20), sequence and structural comparisons (21), or 3D templates (22). In the case of the latter, these methods search between proteins for local structural similarities over a few signature residues that represent the telltale parts of a functional site, so-called "3D templates" (3,14,18,(22)(23)(24). The residue composition of 3D templates is critical, however, and derived from experiments (25) or from analyses of functional sites and determinants (14,15,26). The sensitivity and specificity of template-based annotations still needs to be established experimentally (27, 28), but retrospective controls suggest they often predict enzyme catalytic activity (14,16,17,29,30...

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New Thermophilic and Thermostable Esterase with Sequence Similarity to the Hormone-Sensitive Lipase Family, Cloned from a Metagenomic Library

Cited by 214 publications

References 46 publications

Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples

Insights into bacterial cellulose biosynthesis by functional metagenomics on Antarctic soil samples

From bacterial genomics to metagenomics: concept, tools and recent advances

Prediction and experimental validation of enzyme substrate specificity in protein structures

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