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

Abstract

Hydroxybenzoic 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.

Enzymatic Conversion of CO2: From Natural to Artificial Utilization

“…Single enzyme systems have been developed for the enzymatic Kolbe−Schmitt reaction, 12,145 among them prenylated flavin (prFMN) dependent (de)carboxylases, 146 bivalent metal-dependent (de)carboxylases, and cofactor-independent (de)carboxylases, 10 including a new group of enzymes based on a catalytic dyad mechanism. 147 Furthermore, decarboxylases have been developed for the carboxylation of styrenes, polyaromatics, and heteroaromatic compounds. Recently, their synthetic applications have been reviewed by Leys et al 10 as well as Tomassi et al…”

“…The enzymes are involved in tannin degradation, converting gallic acid and protocatechuic acid to pyrogallol and catechol, respectively (Scheme 22). 147 AGDC1 from Arxula adenivorans and PPP2 from Madurella mycetomatis both form trimers and do not require any organic cofactor. Instead, the enzymes only rely on acid−base catalysis that facilitates the stabilization of the reaction's transition state.…”

“…Instead, the enzymes only rely on acid–base catalysis that facilitates the stabilization of the reaction’s transition state. Each trimer contains one potassium ion, coordinated 3-fold by the Glu88-residue of each monomer, overall forming a distorted octahedral coordination . Although this new group of nonoxidative decarboxylases remains to be investigated for carboxylation reactions, it holds great potential to further expand the substrate scope accessible to carboxylases.…”

Structural Insight into a Yeast Maltase—The BaAG2 from Blastobotrys adeninivorans with Transglycosylating Activity

“…Thus far, only the structure of the gallic acid decarboxylase AGDC1 from Blastobotrys sp. has been determined [12]. Based on the AGDC1 structure, the authors suggested a novel decarboxylation mechanism that combines acid-base catalysis and transition-state stabilization [12].…”

Biochemical and structural characterization of a sphingomonad diarylpropane lyase for cofactorless deformylation

“…Despite its name, some members of the NTF-2-like protein family are catalytic in nature, typically performing dehydration, dehydrochlorination, and more recently decarboxylation, as illustrated in Scheme 1 . Characterized examples of NTF-2-like enzymes include: BaiE, a 3-oxo-Δ 4 -chenodeoxycholyl-CoA dehydratase ( 22 ), scytalone dehydratase (SD) ( 23 ), LinA, a γ-hexachlorocyclohexane dehydrochlorinase ( 24 ), and gallate/protocatechuate decarboxylases (GDC) ( 25 ). Comparison between these systems reveals a common catalytic theme involving a conserved His-Asp dyad that functions as an acid–base catalyst and typically an electron-rich intermediate that is stabilized by an oxyanion hole, often formed by the phenolic group of tyrosine residues.…”