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Methionine-rich loop of multicopper oxidase McoA follows open-to-close transitions with a role in enzyme catalysis

DOI: 10.1021/acscatal.0c01623 DOI Help

Authors: Patricia Borges (Instituto de Tecnologia Quiḿicae Bioloǵica Antońio Xavier, Universidade Nova de Lisboa) , Vânia Brissos (Instituto de Tecnologia Quiḿicae Bioloǵica Antońio Xavier, Universidade Nova de Lisboa) , Guillem Hernandez (Instituto de Tecnologia Quiḿicae Bioloǵica Antońio Xavier, Universidade Nova de Lisboa) , Laura Masgrau (Universitat Autoǹomade Barcelona) , Maria Fátima Lucas (Zymvol Biomodeling) , Emanuele Monza (Zymvol Biomodeling) , Carlos Frazao (Instituto de Tecnologia Quiḿicae Bioloǵica Antońio Xavier, Universidade Nova de Lisboa) , Tiago Cordeiro (Instituto de Tecnologia Quiḿicae Bioloǵica Antońio Xavier, Universidade Nova de Lisboa) , Lígia O. Martins (Instituto de Tecnologia Quiḿicae Bioloǵica Antońio Xavier, Universidade Nova de Lisboa)
Co-authored by industrial partner: Yes

Type: Journal Paper
Journal: Acs Catalysis

State: Published (Approved)
Published: June 2020

Abstract: Multicopper oxidases oxidize a vast range of aromatic substrates coupled to the reduction of molecular oxygen to water. A vast broad spectrum of applications reflects their high biotechnological importance. The crystal structure of McoA from the hyperthermophilic bacteria Aquifex aeolicus has the most tightly compact and hydrophobic core among its prokaryotic counterparts. A 29-residue long loop enriched in glycines and methionines (Met-loop) close to the active T1 Cu center is not detected in the electron density maps. Accurate prediction of loop structures remains challenging, especially for long segments with sizable conformational space. Therefore, a combination of Rosetta and molecular dynamics simulations with ensemble-based small-angle X-ray scattering analysis was used to probe the conformational landscape of the Met-loop. The results indicate a highly flexible omega-loop, which is nevertheless not random but preferentially follows open-to-close transitions, exposing or occluding the T1 Cu site. Loop-truncated variants maintain wild-type stability and consistently lower and higher catalytic efficiencies (kcat/Km) for organic and metal substrates, respectively. Our results suggest that the loop transient dynamic equilibrium can exert important switch-like regulatory function, defining a role for Met-rich motifs as dynamic gate-gappers. This work provides insights into the dynamics of Met-rich loops essential to understand the molecular determinants of substrate promiscuity and catalytic rates within multicopper oxidases. We anticipate that engineering the Met-loop structural dynamics will unleash important changes in enzyme function and specificity with impact on their applications.

Journal Keywords: Laccases; hyperthermophilic enzymes; X-ray; SAXS; Rosetta; methionine-rich; low-complexity region; loop modeling

Diamond Keywords: Enzymes

Subject Areas: Chemistry, Biology and Bio-materials


Instruments: B21-High Throughput SAXS

Other Facilities: BM29 at ESRF

Added On: 18/06/2020 13:57

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Scattering Small Angle X-ray Scattering (SAXS)