Enzymatic synthesis of model substrates recognized by glucuronoyl esterases from Podospora anserina and Myceliophthora thermophila

Abstract

Glucuronoyl esterases (GEs) are recently discovered enzymes that are suggested to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid. Although their potential use for enhanced enzymatic biomass degradation and synthesis of valuable chemicals renders them attractive research targets for biotechnological applications, the difficulty to purify natural fractions of lignin-carbohydrate complexes hampers the characterization of fungal GEs. In this work, we report the synthesis of three aryl alkyl or alkenyl D-glucuronate esters using lipase B from Candida antarctica (CALB) and their use to determine the kinetic parameters of two GEs, StGE2 from the thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophile) and PaGE1 from the coprophilous fungus Podospora anserina. PaGE1 was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter and purified to its homogeneity (63 kDa). The three D-glucuronate esters contain an aromatic UV-absorbing phenol group that facilitates the quantification of their enzymatic hydrolysis by HPLC. Both enzymes were able to hydrolyze the synthetic esters with a pronounced preference towards the cinnamyl-D-glucuronate ester. The experimental results were corroborated by computational docking of the synthesized substrate analogues. We show that the nature of the alcohol portion of the hydrolyzed ester influences the catalytic efficiency of the two GEs.

Functional expression and characterization of a glucuronoyl esterase from the fungus <i>Neurospora crassa</i>: identification of novel consensus sequences containing the catalytic triad

“…These sequences are strongly conserved in all GE sequences, suggesting again the importance of these residues as the components of catalytic triad. In support of our proposal, the crystal structures of Cip2 and StGE (Charavgi et al, 2013;Pokkuluri et al, 2011) showed that the disulfide bond is formed between the cysteine residues equivalent to Cys304 and Cys440 in NcGE that links the consensus sequences VTGCSRXGKGA and HC, hence bringing serine and histidine residues close together in the catalytic site (Charavgi et al, 2013;Katsimpouras et al, 2014). Cys336 involved in another disulfide bond with Cys412 is near E328 and both of these residues are also highly conserved, possibly assisting in introducing glutamic acid to create the catalytic triad.…”

“…Besides, based on the crystal structures of Cip2 (Pokkuluri et al, 2011) and StGE2 (Charavgi et al, 2013), the catalytic triad of GEs was suggested to be composed of two more amino acids, glutamic acid and histidine, located Cterminal to the nucleophile serine. The molecular docking study of StGE2 with the model substrate (Katsimpouras et al, 2014) also supported this idea, which led to the hypothesis that Ser-Glu-His is the standard catalytic triad for GEs. Taking these into consideration, we propose two more consensus sequences constituting the catalytic triad of GEs: PQESG and HC containing the glutamic acid and histidine residues (underlined; position 328 and 439 in NcGE, respectively).…”

“…This consensus was further supported by the crystal structures of two glucuronoyl esterases, Cip2 and StGE2 (Charavgi et al, 2013;Pokkuluri et al, 2011), which confirmed the presence of this sequence in the catalytic center and the role of central serine residue as a nucleophile in the substrate cleavage. Later, the cloning of ScGE from Schizophyllum commune (Wong et al, 2012) was performed, followed by two other GEs from Podospora anserine and Cerrena unicolor (d 'Errico et al, 2015;Katsimpouras et al, 2014).…”

Plant biomass degrading ability of the coprophilic ascomycete fungus Podospora anserina

“…To complete the set of enzymes required for complete degradation of hemicelluloses, two CE members were studied. The methyl glucuronoyl esterase from family CE15 was able to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-D-glucuronic acid (Katsimpouras et al, 2014). The acetyl esterase from family CE16 was versatile in terms of activity on xylan.…”

Microbial Glucuronoyl Esterases: 10 Years after Discovery

“…GEs not only appear to be inducible constituents of plant cell wall-degrading enzyme systems but also are frequently constituents of bi-or multimodular enzymes. Some of them, such as GE from T. reesei and Podospora anserina, contain a family 1 carbohydrate binding module, CBM1 (6,13). Phanerochaete chrysosporium produces two forms, one without and one with the CBM1 module (12).…”