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FEBS JournalVolume 289, Issue 21, November 2022, Pages 6714-6730

Crystal structure of a family VIII β-lactamase fold hydrolase reveals the molecular mechanism for its broad substrate scope(Article)(Open Access)

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  • aIQFR, CSIC, Madrid, Spain
  • bICP, CSIC, Madrid, Spain
  • cPATENT CO, DOO, Mišićevo, Serbia

Abstract

Family VIII esterases present similarities to class C β-lactamases, which show nucleophilic serines located at the S-X-X-K motif instead of the G-X-S-X-G or G-D-S-(L) motif shown by other carboxylesterase families. Here, we report the crystal structure of a novel family VIII (subfamily VIII. I) esterase (EH7; denaturing temperature, 52.6 ± 0.3 °C; pH optimum 7.0–9.0) to deepen its broad substrate range. Indeed, the analysis of the substrate specificity revealed its capacity to hydrolyse nitrocefin as a model chromogenic cephalosporin substrate (40.4 ± 11.4 units·g−1), and a large battery of 66 structurally different esters (up to 1730 min−1), including bis(2-hydroxyethyl)-terephthalate (241.7 ± 8.5 units·g−1) and the mycotoxin T-2 (1220 ± 52 units·g−1). It also showed acyltransferase activity through the synthesis of benzyl 3-oxobutanoate (40.4 ± 11.4 units·g−1) from benzyl alcohol and vinyl acetoacetate. Such a broad substrate scope is rare among family VIII esterases and lipolytic enzymes. Structural analyses of free and substrate-bound forms of this homooctamer esterase suggest that EH7 presents a more opened and exposed S1 site having no steric hindrance for the entrance of substrates to the active site, more flexible R1, R2 and R3 regions allowing for the binding of a wide spectrum of substrates into the active site, and small residues in the conserved motif Y-X-X containing the catalytic Tyr enabling the entrance of large substrates. These unique structural elements in combination with docking experiments allowed us to gain valuable insights into the substrate specificity of this esterase and possible others belonging to family VIII. © 2022 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

Author keywords

esterasemetagenomepromiscuityα/β-fold hydrolaseβ-lactamase

Indexed keywords

EMTREE drug terms:acyltransferasebenzyl alcoholbeta lactamasecarboxylesterasecephalosporinesteresterasefamily VIII beta lactamasehydrolasenitrocefinT 2 toxinunclassified drugesterase
EMTREE medical terms:Articlebiochemistrycrystal structurecrystallizationenzyme active siteenzyme activityenzyme analysisenzyme specificityenzyme substrateGenBankhigh performance liquid chromatographyisoelectric pointmolecular dockingmolecular mechanicsmolecular weightnonhumanpHreaction timeretention timesite directed mutagenesisX ray crystallographychemistrymetabolism
MeSH:beta-LactamasesCarboxylesteraseCatalytic DomainEsterasesSubstrate Specificity

Molecular Sequence Numbers:

GENBANK,

KY483644(referenced)

Chemicals and CAS Registry Numbers:

acyltransferase, 9012-30-0, 9054-54-0; benzyl alcohol, 100-51-6; beta lactamase, 9073-60-3; carboxylesterase, 59536-71-9, 83380-83-0, 9016-18-6, 9028-01-7; cephalosporin, 11111-12-9; CRISPR associated endonuclease Cas9; esterase, 9013-79-0; hydrolase, 9027-41-2; nitrocefin, 41906-86-9; T 2 toxin, 21259-20-1;

beta-Lactamases; Carboxylesterase; Esterases

Funding details

Funding sponsor Funding number Acronym
Ministerio de Ciencia e InnovaciónMICINN
Agencia Estatal de InvestigaciónAEI
Ministerio de Asuntos Económicos y Transformación Digital, Gobierno de EspañaMINECO
European Regional Development FundERDF
Horizon 2020PCIN‐2017‐078,PDC2021‐121534‐I00,PID2020‐112758RB‐I00,PID2019‐105838RB‐C33,604814,BIO2017-85522-R
Ministerio de Educación, Cultura y DeporteFPU17/00044MECD
Horizon 2020 Framework Programme
See opportunities by H2020		101000327H2020
Ministerio de Economía y CompetitividadBES‐2015‐073829MINECO
European Commission
See opportunities by EC		2020AEP061EC

  • 1

    This study was conducted under the auspices of the FuturEnzyme Project funded by the European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No. 101000327. We also acknowledge financial support under Grants PCIN\u20102017\u2010078 (within the Marine Biotechnology ERA\u2010NET, GA No. 604814), BIO2017\u201085522\u2010R (MF), PID2020\u2010112758RB\u2010I00 (MF), PDC2021\u2010121534\u2010I00 (MF), PID2019\u2010105838RB\u2010C31 (FJP) and PID2019\u2010105838RB\u2010C33 (JS\u2010A) from the Ministerio de Econom\u00EDa, Industria y Competitividad, Ministerio de Ciencia e Innovaci\u00F3n, Agencia Estatal de Investigaci\u00F3n (AEI) (Digital Object Identifier 10.13039/501100011033 ), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (\u2018NextGen\u2010erationEU/PRTR\u2019), and Grant 2020AEP061 (MF) from the Agencia Estatal CSIC. C Coscol\u00EDn thanks the Ministerio de Econom\u00EDa y Competitividad and FEDER for a PhD fellowship (Grant BES\u20102015\u2010073829). JLG\u2010A acknowledges support from the Spanish Ministry of Education, Culture and Sport through the National Program FPU (FPU17/00044). The authors acknowledge David Almendral and Rutz Matesanz for supporting the circular dichroism analysis, and Rafael Bargiela for the design of Fig.\u2009 2 . We thank the staff of the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance at the BL13\u2010XALOC beamline. Open access funding enabled and organized by ProjektDEAL.

  • 2

    This study was conducted under the auspices of the FuturEnzyme Project funded by the European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No. 101000327. We also acknowledge financial support under Grants PCIN-2017-078 (within the Marine Biotechnology ERA-NET, GA No. 604814), BIO2017-85522-R (MF), PID2020-112758RB-I00 (MF), PDC2021-121534-I00 (MF), PID2019-105838RB-C31 (FJP) and PID2019-105838RB-C33 (JS-A) from the Ministerio de Econom\u00EDa, Industria y Competitividad, Ministerio de Ciencia e Innovaci\u00F3n, Agencia Estatal de Investigaci\u00F3n (AEI) (Digital Object Identifier 10.13039/501100011033), Fondo Europeo de Desarrollo Regional (FEDER) and the European Union (\u2018NextGen-erationEU/PRTR\u2019), and Grant 2020AEP061 (MF) from the Agencia Estatal CSIC. C Coscol\u00EDn thanks the Ministerio de Econom\u00EDa y Competitividad and FEDER for a PhD fellowship (Grant BES-2015-073829). JLG-A acknowledges support from the Spanish Ministry of Education, Culture and Sport through the National Program FPU (FPU17/00044). The authors acknowledge David Almendral and Rutz Matesanz for supporting the circular dichroism analysis, and Rafael Bargiela for the design of Fig. 2. We thank the staff of the Synchrotron Radiation Source at Alba (Barcelona, Spain) for assistance at the BL13-XALOC beamline. Open access funding enabled and organized by ProjektDEAL.

  • ISSN: 1742464X
  • CODEN: FJEOA
  • Source Type: Journal
  • Original language: English
  • DOI: 10.1111/febs.16554
  • PubMed ID: 35694902
  • Document Type: Article
  • Publisher: John Wiley and Sons Inc

  Sanz-Aparicio, J.; Serrano 119, Madrid, Spain;
  Ferrer, M.; ICP, CSIC, Madrid, Spain;
© Copyright 2023 Elsevier B.V., All rights reserved.

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