The structure of a novel glucuronoyl esterase fromMyceliophthora thermophilagives new insights into its role as a potential biocatalyst

Abstract

The increasing demand for the development of efficient biocatalysts is a consequence of their broad industrial applications. Typical difficulties that are encountered during their exploitation in a variety of processes are interconnected with factors such as temperature, pH, product inhibitors etc. To eliminate these, research has been directed towards the identification of new enzymes that would comply with the required standards. To this end, the recently discovered glucuronoyl esterases (GEs) are an enigmatic family within the carbohydrate esterase (CE) family. Structures of the thermophilic StGE2 esterase from Myceliophthora thermophila (synonym Sporotrichum thermophile), a member of the CE15 family, and its S213A mutant were determined at 1.55 and 1.9 Å resolution, respectively. The first crystal structure of the S213A mutant in complex with a substrate analogue, methyl 4-O-methyl-β-D-glucopyranuronate, was determined at 2.35 Å resolution. All of the three-dimensional protein structures have an α/β-hydrolase fold with a three-layer αβα-sandwich architecture and a Rossmann topology and comprise one molecule per asymmetric unit. These are the first crystal structures of a thermophilic GE both in an unliganded form and bound to a substrate analogue, thus unravelling the organization of the catalytic triad residues and their neighbours lining the active site. The knowledge derived offers novel insights into the key structural elements that drive the hydrolysis of glucuronic acid esters.

Microbial Glucuronoyl Esterases: 10 Years after Discovery

“…The twisted ␤-sheet is sandwiched between two layers of ␣-helices with the catalytic triad Ser-His-Glu exposed on the protein surface. A similar 3D structure was reported for GE from Myceliophthora thermophila (earlier assigned as Sporotrichum thermophile) (20) (Fig. 1).…”

“…A mutation of catalytic serine to alanine in M. thermophila GE abolished the enzyme activity (21) but did not affect its active site architecture (20). The mutant was also successfully crystallized with methyl 4-O-methyl-D-glucopyranuronate, an artificial GE substrate (5), to reveal the topology of the active site (20). In contrast to the majority of serine type esterases, glutamic acid replaces aspartic acid in known 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.…”

“…From the alignment of homologous sequences, a novel consensus sequence G-C-S-R-X-G conserved in CE15 family members was suggested (Topakas et al, 2010). 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).…”

“…In this paper, using the sequences of seven characterized GEs, we propose that this motif could be expanded to VTGCSRXGKGA (the original motif is underlined). 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.…”