Crystal Structure and Biochemical Properties of a Novel Thermostable Esterase Containing an Immunoglobulin-Like Domain

Levisson, Mark; Sun, Lei; Hendriks, Sjon; Swinkels, Peter; Akveld, Twan; Bultema, Jelle B.; Barendregt, Arjan; Heuvel, Robert H. H. van den; Dijkstra, Bauke W.; Oost, John van der; Kengen, Servé W. M.

doi:10.1016/j.jmb.2008.10.075

Cited by 36 publications

(36 citation statements)

References 40 publications

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“…As determined by Matcher (EMBOSS package), among Lake Arreo ␣/␤ hydrolases, LAE4 and LAE7 were the most similar enzymes (71.4% sequence identity), whereas LAE5 and LAE6 were the most different at the sequence level (18.2% identity) (see Table S3 in the supplemental material). The LAE1 protein was structurally most similar to thermophilic esterase from Thermotoga maritima (31%; Protein Data Bank [PDB] code 3DOH_A [42]) and ␣/␤ hydrolase from Agrobacterium tumefaciens (24%; PDB code 2r8b); LAE2 was most similar to GDSL-like brain platelet-activating factor acetylhydrolases (SGNH_hydrolase subfamily) (31%, PDB code 1FXW [43], and 28%, PDB code 1ES9 [44]); LAE3 was most similar to thermostable esterase/lipase from uncultured bacterium (36%; PDB code 3V9A_A) and hormone-sensitive esterase (35%; PDB code 3fak [45]); LAE4 was most similar to esterase from Pyrobaculum calidifontis (25%; PDB code 2YH2_A [46]) and bacterial ␣/␤ hydrolase (19%; PDB code 3k2i); LAE5 was most similar to the GDSL family lipolytic protein (SGNH_hydrolase subfamily) from Alicyclobacillus acidocaldarius subsp. acidocaldarius Dsm 446 (54%; PDB code 3RJT_B); LAE6 was most similar to thermophilic and thermostable carboxylesterase from the metagenomic library (41%; PDB code 2c7b [47]); and LAE7 was most similar to GDSLlike brain platelet-activating factor acetylhydrolase (SGNH_hy-drolase subfamily) (32%; PDB code 1ES9 [44]).…”

Section: Resultsmentioning

confidence: 99%

Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

Martínez-Martínez

Alcaide

Tchigvintsev

et al. 2013

Appl Environ Microbiol

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The esterases and lipases from the ␣/␤ hydrolase superfamily exhibit an enormous sequence diversity, fold plasticity, and activities. Here, we present the comprehensive sequence and biochemical analyses of seven distinct esterases and lipases from the metagenome of Lake Arreo, an evaporite karstic lake in Spain (42°46=N, 2°59=W; altitude, 655 m). Together with oligonucleotide usage patterns and BLASTP analysis, our study of esterases/lipases mined from Lake Arreo suggests that its sediment contains moderately halophilic and cold-adapted proteobacteria containing DNA fragments of distantly related plasmids or chromosomal genomic islands of plasmid and phage origins. This metagenome encodes esterases/lipases with broad substrate profiles (tested over a set of 101 structurally diverse esters) and habitat-specific characteristics, as they exhibit maximal activity at alkaline pH (8.0 to 8.5) and temperature of 16 to 40°C, and they are stimulated (1.5 to 2.2 times) by chloride ions (0.1 to 1.2 M), reflecting an adaptation to environmental conditions. Our work provides further insights into the potential significance of the Lake Arreo esterases/lipases for biotechnology processes (i.e., production of enantiomers and sugar esters), because these enzymes are salt tolerant and are active at low temperatures and against a broad range of substrates. As an example, the ability of a single protein to hydrolyze triacylglycerols, (non)halogenated alkyl and aryl esters, cinnamoyl and carbohydrate esters, lactones, and chiral epoxides to a similar extent was demonstrated. E sterases and lipases from the ␣/␤ hydrolase family have received considerable attention, because they are widely distributed within the microbial communities operating in most of environments where they have important physiological functions (1) and because they are one of the most important groups of biocatalysts for biotechnological applications (2-4). Upon searching the list of genes using Pfam (the protein family database [5]) from the approximately 140 metagenomic projects in various stages of sequencing on the GOLD website (Genomes OnLine Database; http://www.genomesonline.org/) and the available sequences of esterases and lipases, more than 72,000 predicted esterases/lipases of the ␣/␤ hydrolase superfamily were retrieved, which revealed the richness of uncultured biodiversity (6), to provide wide collections of such biocatalysts. This is one of the largest protein families with available sequences. In relation to the cultivation-independent methods used to identify them, it should be highlighted that sequence-based metagenomics only provide the presumptive compositional and functional blueprint represented in the community genome (7,8), but at the same time, this method causes serious problems regarding both sequencing errors (9) and the erroneous assignment of substrate specificity (10). In contrast, the activity-directed techniques have been shown to provide a direct view of known or new protein families and functionalities (for examples, see referen...

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Section: Resultsmentioning

confidence: 99%

Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

Martínez-Martínez

Alcaide

Tchigvintsev

et al. 2013

Appl Environ Microbiol

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“…This activity level was lower than those of EstA and AXE from Thermotoga maritima, which displayed optimal esterase activity at or above 95°C and 100°C, respectively (10,11). However, 7-ACA was not considered their natural substrate, because the stability of 7-ACA at the optimal growth temperature (80°C) is very low (10,11). Moreover, EstD1 was stable over a broad pH range (4.0 to 11.0), one that is broader than those of AXE from B. subtilis CICC 20034 (pH 6.0 to 11.0) (5), EstA from Thermotoga maritima (pH 5.0 to 11.0) (11), and AXE from Thermotoga maritima (pH 4.8 to 9.2) (10), and it even displayed activity when the pH reached 12.0 (Fig.…”

Section: Discussionmentioning

confidence: 81%

“…3D). This activity level was lower than those of EstA and AXE from Thermotoga maritima, which displayed optimal esterase activity at or above 95°C and 100°C, respectively (10,11). However, 7-ACA was not considered their natural substrate, because the stability of 7-ACA at the optimal growth temperature (80°C) is very low (10,11).…”

Section: Discussionmentioning

confidence: 92%

“…Thus far, B. subtilis (5,6,8,13,15), B. pumilus PS213 (7), B. halodurans C-125 (12), Thermotoga maritima MSB8 (10,11), and Thermoanaerobacterium saccharolyticum JW/SL-YS485 (9) species have been reported to have deacetylase activity toward 7-ACA. However, only a few enzymes involved in this process have been characterized, including AXE from B. subtilis CICC 20034 (5), CAH from B. subtilis SHS 0133 (6), Axe from B. pumilus PS213 (7), Cah from B. subtilis subsp.…”

Section: Discussionmentioning

confidence: 99%

“…Among these characterized proteins, only 6 have been demonstrated to have 7-ACA deacetylase activity, including acetyl xylan esterase (AXE) from Bacillus subtilis CICC 20034 (5), CAH from B. subtilis SHS 0133 (6), Axe from Bacillus pumilus PS213 (7), Cah from B. subtilis subsp. subtilis strain 168 (8), Axe1 from Thermoanaerobacterium saccharolyticum JW/SL-YS485 (9), and TM0077 from Thermotoga maritima MSB8 (10,11). In the CE-12 family, only 11 sequences have been characterized among the 570 sequences sub-mitted.…”

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confidence: 99%

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Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis

Ding

Han

et al. 2016

J Bacteriol

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Deacetylation of 7-aminocephalosporanic acid (7-ACA) at position C-3 provides valuable starting material for producing semisynthetic ␤-lactam antibiotics. However, few enzymes have been characterized in this process before now. Comparative analysis of the genome of the thermophilic bacterium Alicyclobacillus tengchongensis revealed a hypothetical protein (EstD1) with typical esterase features. The EstD1 protein was functionally cloned, expressed, and purified from Escherichia coli BL21(DE3). It indeed displayed esterase activity, with optimal activity at around 65°C and pH 8.5, with a preference for esters with short-chain acyl esters (C 2 to C 4 ). Sequence alignment revealed that EstD1 is an SGNH hydrolase with the putative catalytic triad Ser15, Asp191, and His194, which belongs to carbohydrate esterase family 12. EstD1 can hydrolyze acetate at the C-3 position of 7-aminocephalosporanic acid (7-ACA) to form deacetyl-7-ACA, which is an important starting material for producing semisynthetic ␤-lactam antibiotics. EstD1 retained more than 50% of its initial activity when incubated at pH values ranging from 4 to 11 at 65°C for 1 h. To the best of our knowledge, this enzyme is a new SGNH hydrolase identified from thermophiles that is able to hydrolyze 7-ACA. IMPORTANCEDeacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic ␤-lactam antibiotics. These compounds are derived mainly from 7-ACA, which is obtained by chemical or enzymatic processes from cephalosporin C. Enzymatic transformation of 7-ACA is the main method because of the adverse effects chemical deacylation brought to the environment. SGNH hydrolases are widely distributed in plants. However, the tools for identifying and characterizing SGNH hydrolases from bacteria, especially from thermophiles, are rather limited. Here, our work demonstrates that EstD1 belongs to the SGNH family and can hydrolyze acetate at the C-3 position of 7-ACA. Moreover, this study can enrich our understanding of the functions of these enzymes from this family. D eacetyl cephalosporins are highly valuable building blocks for the industrial production of various kinds of semisynthetic ␤-lactam antibiotics. These compounds are derived mainly from 7-aminocephalosporanic acid (7-ACA), which is obtained from cephalosporin C by chemical or enzymatic processes (1). Environmentally friendly enzymatic processes are widely used because of the advantages of mild reaction conditions, such as pH, temperature, and high yield. In many cases, chemical modifications can be made at the C-3= position of 7-ACA by nucleophilic displacement of the acetate group; the removal of the acetate group is necessary to make other modifications at this position. The enzyme cephalosporin C deacetylase (CCD; also called acetylesterase) catalyzes the hydrolysis of acetate from the 3= position of 7-ACA. These enzyme activities have been found in citrus peels (2) and mammalian tissues, where it appears to be most prevalent in the liver and k...

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Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6

Zhu

Zheng

et al. 2015

J. Basic Microbiol.

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A new lipolytic enzyme gene was cloned from a thermophile Geobacillus sp. JM6. The gene contained 750 bp and encoded a 249-amino acid protein. The recombinant enzyme was expressed and purified from Escherichia coli BL21 (DE3) with a molecular mass of 33.6 kDa. Enzyme assays using p-nitrophenyl esters with different acyl chain lengths as the substrates confirmed its esterase activity, yielding the highest activity with p-nitrophenyl butyrate. When p-nitrophenyl butyrate was used as a substrate, the optimum reaction temperature and pH for the enzyme were 60 °C and pH 7.5, respectively. Geobacillus sp. JM6 esterase showed excellent thermostability with 68% residual activity after incubation at 100 °C for 18 h. A theoretical structural model of strain JM6 esterase was developed with a monoacylglycerol lipase from Bacillus sp. H-257 as a template. The predicted core structure exhibits an α/β hydrolase fold, and a putative catalytic triad (Ser97, Asp196, and His226) was identified. Inhibition assays with PMSF indicated that serine residue is involved in the catalytic activity of strain JM6 esterase. The recombinant esterase showed a relatively good tolerance to the detected detergents and denaturants, such as SDS, Chaps, Tween 20, Tween 80, Triton X-100, sodium deoxycholate, urea, and guanidine hydrochloride.

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Crystal Structure and Biochemical Properties of a Novel Thermostable Esterase Containing an Immunoglobulin-Like Domain

Cited by 36 publications

References 40 publications

Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

Biochemical Diversity of Carboxyl Esterases and Lipases from Lake Arreo (Spain): a Metagenomic Approach

Identification and Characterization of a New 7-Aminocephalosporanic Acid Deacetylase from Thermophilic Bacterium Alicyclobacillus tengchongensis

Molecular cloning and characterization of a new and highly thermostable esterase from Geobacillus sp. JM6

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