From a metagenomic source to a high-resolution structure of a novel alkaline esterase

Pereira, Mariana Rangel; Maester, Thaís Carvalho; Mercaldi, Gustavo Fernando; Lemos, Eliana Gertrudes de Macedo; Hyvönen, Marko; Balan, Andrea

doi:10.1007/s00253-017-8226-4

Cited by 36 publications

(34 citation statements)

References 46 publications

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“…20 Recently, Pereira et al have characterized a mesophilic esterase, Est8, having higher catalytic efficiency against short-chain esters with mild thermal stability. 26 Sequence alignment showed the presence of Tyr near the catalytic site of Est8 (Figure 1a, upper panel), further supporting the idea that the presence of Tyr can be attributed to high enzymatic activity. 44…”

Section: Discussionsupporting

confidence: 58%

“…Mesophilic esterases; rPPE ( Pseudomonas putida ) 21,25 and Est8 (metagenome-derived). 26 Hyperthermophilic esterases; EstE1 (thermal environmental sample) 27,28 and Est2 ( Alicyclobacillus acidocaldarius ). 29 Psychrophilic esterases; EstSP1 ( Sphingomonas glacialis PAMC 26605), 30 EstS ( Shewanella halifaxensis ), 31 and EstG ( Sphingobium sp.).…”

Section: Introductionmentioning

confidence: 99%

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Roles of Active-Site Aromatic Residues in Cold Adaptation of Sphingomonas glacialis Esterase EstSP1

et al. 2017

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The aromatic amino acids, Tyr or Trp, which line the active-site walls of esterases, stabilize the catalytic His loop via hydrogen bonding. A Tyr residue is preferred in extremophilic esterases (psychrophilic or hyperthermophilic esterases), whereas a Trp residue is preferred in moderate-temperature esterases. Here, we provide evidence that Tyr and Trp play distinct roles in cold adaptation of the psychrophilic esterase EstSP1 isolated from an Arctic bacterium Sphingomonas glacialis PAMC 26605. Stern–Volmer plots showed that the mutation of Tyr191 to Ala, Phe, Trp, and His resulted in reduced conformational flexibility of the overall protein structure. Interestingly, the Y191W and Y191H mutants showed increased thermal stability at moderate temperatures. All Tyr191 mutants showed reduced catalytic activity relative to wild-type EstSP1. Our results indicate that Tyr with its phenyl hydroxyl group is favored for increased conformational flexibility and high catalytic activity of EstSP1 at low temperatures at the expense of thermal stability. The results of this study suggest that, in the permanently cold Arctic zone, enzyme activity has been selected for psychrophilic enzymes over thermal stability. The results presented herein provide novel insight into the roles of Tyr and Trp residues for temperature adaptation of enzymes that function at low, moderate, and high temperatures.

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Section: Discussionsupporting

confidence: 58%

Section: Introductionmentioning

confidence: 99%

Roles of Active-Site Aromatic Residues in Cold Adaptation of Sphingomonas glacialis Esterase EstSP1

et al. 2017

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“…The ability of the enzyme to bind PEG molecule is attributed to a protruding cavity consisting of mostly hydrophobic residues. The size of the cavity correlates with the substrate specificity of the enzymes of this class in terms of size of the preferable substrates [54]. The length of PMGL2 active site cavity correlates with spatial parameters of middle-chain substrates (C8 and C10) which are specifically preferred by PMGL2 [21].…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine

et al. 2020

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Lipases comprise a large class of hydrolytic enzymes which catalyze the cleavage of the ester bonds in triacylglycerols and find numerous biotechnological applications. Previously, we have cloned the gene coding for a novel esterase PMGL2 from a Siberian permafrost metagenomic DNA library. We have determined the 3D structure of PMGL2 which belongs to the hormone-sensitive lipase (HSL) family and contains a new variant of the active site motif, GCSAG. Similar to many other HSLs, PMGL2 forms dimers in solution and in the crystal. Our results demonstrated that PMGL2 and structurally characterized members of the GTSAG motif subfamily possess a common dimerization interface that significantly differs from that of members of the GDSAG subfamily of known structure. Moreover, PMGL2 had a unique organization of the active site cavity with significantly different topology compared to the other lipolytic enzymes from the HSL family with known structure including the distinct orientation of the active site entrances within the dimer and about four times larger size of the active site cavity. To study the role of the cysteine residue in GCSAG motif of PMGL2, the catalytic properties and structure of its double C173T/C202S mutant were examined and found to be very similar to the wild type protein. The presence of the bound PEG molecule in the active site of the mutant form allowed for precise mapping of the amino acid residues forming the substrate cavity.

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“…The catalytic triads of WDEst17 and PHE21 are formed by Ser 94 ‐Asp 200 ‐His 228 and Ser 164 ‐Asp 260 ‐His 290 , respectively (Figures S8 and S9). The differences in protein sequences and structures of the two enzymes should be the main reasons leading to the different stereo‐preference . However, just like we cannot predict the detailed characteristics of one enzyme simple based upon protein sequence analysis and structure analysis, we cannot predict the stereo‐preference and the stereo‐selectivity of one enzyme, at least at current stage.…”

Section: Discussionmentioning

confidence: 99%

“…The differences in protein sequences and structures of the two enzymes should be the main reasons leading to the different stereo-preference. 27,28 However, just like we cannot predict the detailed characteristics of one enzyme simple based upon protein sequence analysis and structure analysis, we cannot predict the stereo-preference and the stereo-selectivity of one enzyme, at least at current stage. Next, the studies of the crystals of WDEst17 and PHE21 and the interaction of the two esterases with the two enantiomers of (±)ethyl 3-hydroxybutyrate may provide more information for the explanation of opposite stereo-preference.…”

Section: Discussionmentioning

confidence: 99%

Functional characterization of salt‐tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)‐3‐hydroxybutyrate

Wang

Zhang

et al. 2018

Chirality

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The two enantiomers of ethyl 3-hydroxybutyrate are important intermediates for the synthesis of a great variety of valuable chiral drugs. The preparation of chiral drug intermediates through kinetic resolution reactions catalyzed by esterases/lipases has been demonstrated to be an efficient and environmentally friendly method. We previously functionally characterized microbial esterase PHE21 and used PHE21 as a biocatalyst to generate optically pure ethyl (S)-3-hydroxybutyrate. Herein, we also functionally characterized one novel salt-tolerant microbial esterase WDEst17 from the genome of Dactylosporangium aurantiacum subsp. Hamdenensis NRRL 18085. Esterase WDEst17 was further developed as an efficient biocatalyst to generate (R)-3-hydroxybutyrate, an important chiral drug intermediate, with the enantiomeric excess being 99% and the conversion rate being 65.05%, respectively, after process optimization. Notably, the enantio-selectivity of esterase WDEst17 was opposite than that of esterase PHE21. The identification of esterases WDEst17 and PHE21 through genome mining of microorganisms provides useful biocatalysts for the preparation of valuable chiral drug intermediates.

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From a metagenomic source to a high-resolution structure of a novel alkaline esterase

Cited by 36 publications

References 46 publications

Roles of Active-Site Aromatic Residues in Cold Adaptation of Sphingomonas glacialis Esterase EstSP1

Roles of Active-Site Aromatic Residues in Cold Adaptation of Sphingomonas glacialis Esterase EstSP1

Crystal structure of PMGL2 esterase from the hormone-sensitive lipase family with GCSAG motif around the catalytic serine

Functional characterization of salt‐tolerant microbial esterase WDEst17 and its use in the generation of optically pure ethyl (R)‐3‐hydroxybutyrate

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