Rational Enzyme Engineering Through Biophysical and Biochemical Modeling

Sandra Acebes; Elena Fernandez-Fueyo; Emanuele Monza; M. Fatima Lucas; David Almendral; Francisco J. Ruiz-Dueñas; Henrik Lund; Angel T. Martinez; Victor Guallar

doi:10.1021/acscatal.6b00028

Rational Enzyme Engineering Through Biophysical and Biochemical Modeling

ACS Catalysis ◽

10.1021/acscatal.6b00028 ◽

2016 ◽

Vol 6 (3) ◽

pp. 1624-1629 ◽

Cited By ~ 25

Author(s):

Sandra Acebes ◽

Elena Fernandez-Fueyo ◽

Emanuele Monza ◽

M. Fatima Lucas ◽

David Almendral ◽

...

Keyword(s):

Enzyme Engineering ◽

Biochemical Modeling

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Super-precise CRISPR tool enhanced by enzyme engineering

Nature ◽

10.1038/d41586-020-00340-w ◽

2020 ◽

Author(s):

Heidi Ledford

Keyword(s):

Enzyme Engineering

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Enzyme Engineering and Evolution: General Methods

10.1016/s0076-6879(20)x0015-2 ◽

2020 ◽

Keyword(s):

Enzyme Engineering

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Accelerating the Rate-Limiting Step in Novel Enzymatic Carbohydrate-to-Hydrogen Technology by Enzyme Engineering

10.21236/ada567201 ◽

2011 ◽

Author(s):

Y. P. Zhang

Keyword(s):

Enzyme Engineering ◽

Rate Limiting Step ◽

Limiting Step ◽

Hydrogen Technology ◽

Rate Limiting

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Crystal structures of non-oxidative decarboxylases reveal a new mechanism of action with a catalytic dyad and structural twists

Scientific Reports ◽

10.1038/s41598-021-82660-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Matthias Zeug ◽

Nebojsa Markovic ◽

Cristina V. Iancu ◽

Joanna Tripp ◽

Mislav Oreb ◽

...

Keyword(s):

Crystal Structures ◽

Active Site ◽

Enzyme Engineering ◽

Base Catalysis ◽

Oxidative Decarboxylation ◽

Transition State Stabilization ◽

Major Bottleneck ◽

Hydroxybenzoic Acids ◽

Potassium Binding ◽

Β Sheet

AbstractHydroxybenzoic acids, like gallic acid and protocatechuic acid, are highly abundant natural compounds. In biotechnology, they serve as critical precursors for various molecules in heterologous production pathways, but a major bottleneck is these acids’ non-oxidative decarboxylation to hydroxybenzenes. Optimizing this step by pathway and enzyme engineering is tedious, partly because of the complicating cofactor dependencies of the commonly used prFMN-dependent decarboxylases. Here, we report the crystal structures (1.5–1.9 Å) of two homologous fungal decarboxylases, AGDC1 from Arxula adenivorans, and PPP2 from Madurella mycetomatis. Remarkably, both decarboxylases are cofactor independent and are superior to prFMN-dependent decarboxylases when heterologously expressed in Saccharomyces cerevisiae. The organization of their active site, together with mutational studies, suggests a novel decarboxylation mechanism that combines acid–base catalysis and transition state stabilization. Both enzymes are trimers, with a central potassium binding site. In each monomer, potassium introduces a local twist in a β-sheet close to the active site, which primes the critical H86-D40 dyad for catalysis. A conserved pair of tryptophans, W35 and W61, acts like a clamp that destabilizes the substrate by twisting its carboxyl group relative to the phenol moiety. These findings reveal AGDC1 and PPP2 as founding members of a so far overlooked group of cofactor independent decarboxylases and suggest strategies to engineer their unique chemistry for a wide variety of biotechnological applications.

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