Single residues dictate the co-evolution of dual esterases: MCP hydrolases from the α/β hydrolase family

Alcaide, María; Tornes, J.; Stogios, P.J.; Xu, Xiaohui; Gertler, Christoph; Leo, Rosa Di; Bargiela, Rafael; Lafraya, Álvaro; Guazzaroni, María‐Eugenia; López-Cortés, Nieves; Chernikova, Tatyana N.; Golyshina, Olga V.; Nechitaylo, Taras Y.; Plumeier, Iris; Pieper, Dietmar H.; Yakimov, Michail M.; Savchenko, Alexei; Golyshin, Peter N.; Ferrer, Manuel

doi:10.1042/bj20130552

Cited by 31 publications

(40 citation statements)

References 47 publications

(37 reference statements)

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“…If not otherwise stated, 1 mM pNP-propionate was used as the standard assay substrate for the determination of the conditions under which each enzyme displayed activity; pH values between 4.5 and 9.0 (at the optimal temperature), temperatures between 4 and 80°C (using 50 mM Tris-HCl buffer, pH 8.0), and NaCl, KCl, and MgCl 2 concentrations of up to 4 M (using 50 mM Tris-HCl buffer, pH 8.0, and optimal temperatures) were tested. The buffers used to determine the optimal pH for each enzyme have been described previously (3,27).…”

Section: Methodsmentioning

confidence: 99%

“…1) (19). Total DNA was extracted from the gill chambers of the collected specimens as previously described (19); from this DNA, a large-insert pCCFOS1 fosmid library was generated using the Escherichia coli EPI300-T1 R strain (Epicentre Biotechnologies; Madison, WI, USA), and the library was scored for the ability to hydrolyze ␣-naphthyl acetate and tributyrin (27,30). Positive clones were selected, and their DNA inserts were sequenced using a Roche 454 GS FLX Ti sequencer (454 Life Sciences, Branford, CT, USA) at Life Sequencing SL (Valencia, Spain) or were completely Sanger sequenced using universal primers and subsequent primer walking.…”

Section: Methodsmentioning

confidence: 99%

“…Carboxyl esterase activity was assayed using p-nitrophenyl (pNP) esters (read at 410 nm) and structurally diverse esters other than pNP esters (read at 540 nm) in 96-well plates as previously described (27). Unless stated otherwise, standard assay reactions were conducted by adding 2 l of the 2 mg ml Ϫ1 protein stock solution to an assay mixture containing 2 l of ester stock solution (100 mM in acetone [for pNP esters] or acetonitrile [for other esters]) in 196 l of 50 mM Tris-HCl buffer, pH 8.0 (for pNP esters), or 5 mM N-(2-hydroxyethyl) piperazine-N=-(3-propanesulfonic acid) (EPPS) buffer, pH 8.0 (for other esters).…”

Section: Methodsmentioning

confidence: 99%

“…These chemicals included 11 model esters (7 pNP esters and 4 triacylglycerols) (Fig. 4), as well as a battery of 120 structurally different esters (3,6,27,28,37,53) (Fig. 5).…”

Section: Metagenome Library Construction and Screening For Carboxyl Ementioning

confidence: 99%

“…Therefore, understanding the characteristics of other deep-sea enzymes, particularly esterases for which multiple biochemical data are available compared to other deep-sea enzymes (3), might help in understanding the phenomenon of protein versatility and adaptability to different polyextremes. Second, esterases were selected because they are ubiquitous enzymes that are widespread in nature (at least one per genome), multiple commercial preparations that are used at industrial scale are available, and biochemical data of such enzymes from a number of marine habitats (3,27,28) have been reported recently, which collectively may assist to perform an extensive comparative analysis. Finally, they are of great interest as biocatalysts for chemical synthesis (29).…”

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confidence: 99%

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Identification and Characterization of Carboxyl Esterases of Gill Chamber-Associated Microbiota in the Deep-Sea Shrimp Rimicaris exoculata by Using Functional Metagenomics

Alcaide

Tchigvintsev

Martínez-Martínez

et al. 2015

Appl Environ Microbiol

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iThe shrimp Rimicaris exoculata dominates the fauna in deep-sea hydrothermal vent sites along the Mid-Atlantic Ridge (depth, 2,320 m). Here, we identified and biochemically characterized three carboxyl esterases from microbial communities inhabiting the R. exoculata gill that were isolated by naive screens of a gill chamber metagenomic library. These proteins exhibit low to moderate identity to known esterase sequences (<52%) and to each other (11.9 to 63.7%) and appear to have originated from unknown species or from genera of Proteobacteria related to Thiothrix/Leucothrix (MGS-RG1/RG2) and to the Rhodobacteraceae group (MGS-RG3). A library of 131 esters and 31 additional esterase/lipase preparations was used to evaluate the activity profiles of these enzymes. All 3 of these enzymes had greater esterase than lipase activity and exhibited specific activities with ester substrates (<356 U mg ؊1 ) in the range of similar enzymes. MGS-RG3 was inhibited by salts and pressure and had a low optimal temperature (30°C), and its substrate profile clustered within a group of low-activity and substrate-restricted marine enzymes. In contrast, MGS-RG1 and MGS-RG2 were most active at 45 to 50°C and were salt activated and barotolerant. They also exhibited wider substrate profiles that were close to those of highly active promiscuous enzymes from a marine hydrothermal vent (MGS-RG2) and from a cold brackish lake (MGS-RG1). The data presented are discussed in the context of promoting the examination of enzyme activities of taxa found in habitats that have been neglected for enzyme prospecting; the enzymes found in these taxa may reflect distinct habitat-specific adaptations and may constitute new sources of rare reaction specificities. Metagenomics provides a means for the discovery of entirely new enzymes in microorganisms and their communities without the need to culture these microorganisms as individual species, which is technically very difficult (1-5). Although the discovery of new enzyme activities has progressed considerably (6), it has not managed to go beyond the effective identification of enzymatic activities at a rather limited number of environmental sites. While an extensive search in the specialized literature and public databases indicated that microbial communities from approximately 1,800 different sites worldwide have been examined for their genomic content, only in approximately 200 of those locations (11% of the total) have new active clones or enzymes been identified and/or partially characterized. Thus, only a tiny fraction of the Earth's biosphere has been explored for the purpose of enzyme discovery, despite the fact that natural microbial diversity has been recognized to be the major source of new microbes (7). Such diversity also contains novel genes and functions (8) whose activities remain mostly unknown but whose potential to contribute to an innovation-based economy is recognized (9, 10).One of the habitats less explored to date in terms of examining enzyme repertoires is the deep-sea realm, where...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…These chemicals included 11 model esters (7 pNP esters and 4 triacylglycerols) (Fig. 4), as well as a battery of 120 structurally different esters (3,6,27,28,37,53) (Fig. 5).…”

Section: Metagenome Library Construction and Screening For Carboxyl Ementioning

confidence: 99%

mentioning

confidence: 99%

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