Inter-conversion of catalytic abilities in a bifunctional carboxyl/feruloyl-esterase from earthworm gut metagenome.
Carboxyl esterases (CE) exhibit various reaction specificities despite of their overall structural similarity. In present study we have exploited functional metagenomics, saturation mutagenesis and experimental protein evolution to explore residues that have a significant role in substrate discrimination. We used an enzyme, designated 3A6, derived from the earthworm gut metagenome that exhibits CE and feruloyl esterase (FAE) activities with p-nitrophenyl and cinnamate esters, respectively, with a [(k(cat)/K(m))](CE)/[(k(cat)/K(m))](FAE) factor of 17. Modelling-guided saturation mutagenesis at specific hotspots (Lys(281), Asp(282), Asn(316) and Lys(317)) situated close to the catalytic core (Ser(143)/Asp(273)/His(305)) and a deletion of a 34-AA-long peptide fragment yielded mutants with the highest CE activity, while cinnamate ester bond hydrolysis was effectively abolished. Although, single to triple mutants with both improved activities (up to 180-fold in k(cat)/K(m) values) and enzymes with inverted specificity ((k(cat)/K(m))(CE)/(k(cat)/K(m))(FAE) ratio of ∼0.4) were identified, no CE inactive variant was found. Screening of a large error-prone PCR-generated library yielded by far less mutants for substrate discrimination. We also found that no significant changes in CE activation energy occurs after any mutation (7.3 to -5.6 J mol(-1)), whereas a direct correlation between loss/gain of FAE function and activation energies (from 33.05 to -13.7 J mol(-1)) was found. Results suggest that the FAE activity in 3A6 may have evolved via introduction of a limited number of 'hot spot' mutations in a common CE ancestor, which may retain the original hydrolytic activity due to lower restrictive energy barriers but conveys a dynamic energetically favourable switch of a second hydrolytic reaction.
CSIC, Institute of Catalysis, 28049 Madrid, Spain. CSIC, Instituto de Agroquímica y Tecnología de Alimentos, 46980 Valencia, Spain.
José María
JM
Vieites
Azam
A
Ghazi
Ana
A
Beloqui
Julio
J
Polaina
José M
JM
Andreu
Olga V
OV
Golyshina
Taras Y
TY
Nechitaylo
Agnes
A
Waliczek
Michail M
MM
Yakimov
Peter N
PN
Golyshin
Manuel
M
Ferrer
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002265:Carboxylic Ester Hydrolases / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D041981:Gastrointestinal Tract / Q000201:enzymology; D007700:Kinetics; D054892:Metagenome; D016296:Mutagenesis; D009835:Oligochaeta / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase
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