Abstract
The family 15 carbohydrate esterase (CE15) MZ0003, which derives from a marine Arctic metagenome, has a broader substrate scope than other members of this family. Here we report the crystal structure of MZ0003, which reveals that residues comprising the catalytic triad differ from previously-characterized fungal homologs, and resolves three large loop regions that are unique to this bacterial sub-clade. The catalytic triad of the bacterial CE15, which includes Asp 332 as its third member, closely resembles that of family 1 carbohydrate esterases (CE1), despite the overall lower structural similarity with members of this family. Two of the three loop regions form a subdomain that deepens the active site pocket and includes several basic residues that contribute to the high positive charge surrounding the active site. Docking simulations predict specific interactions with the sugar moiety of glucuronic-acid substrates, and with aromatically-substituted derivatives that serve as model compounds for the lignin-carbohydrate complex of plant cell walls. Molecular dynamics simulations indicate considerable flexibility of the sub-domain in the substrate-bound form, suggesting plasticity to accommodate different substrates is possible. The findings from this first reported structure of a bacterial member of the CE15 family provide insight into the basis of its broader substrate specificity.
Highlights
The CAZymes family 15 carbohydrate esterases (CE15s) include members with glucuronoyl esterase activity that are predicted to act on the ester linkages between the 4-O-methyl-glucuronoyl substitutions on xylan of hemicellulose and the aromatic alcohol of lignin as their natural substrate; the so-called lignin-carbohydrate-complex (LCC)
The asymmetric unit of the MZ0003 crystal structure contains only one molecule, a dimer can be generated based on crystallographic symmetry, yielding a quaternary structure resembling that observed in the homolog 3pic (Supplementary Figure 1)
The electron density is well defined in this region, demonstrating that this is not an artifact of poorly defined density due to flexibility. The possibility that this quaternary structure is induced under crystallization can, not be ruled out, as MZ0003 was previously indicated by both gel filtration and native PAGE to be a monomer with extended conformation in solution[9]
Summary
The CAZymes family 15 carbohydrate esterases (CE15s) include members with glucuronoyl esterase activity that are predicted to act on the ester linkages between the 4-O-methyl-glucuronoyl substitutions on xylan of hemicellulose and the aromatic alcohol of lignin as their natural substrate; the so-called lignin-carbohydrate-complex (LCC). Two crystal structures of CE15s, both from fungi, are available to date: the catalytic domain of the Cip[2] enzyme from Hypocrea jecorina (synonym Trichoderma reesei, PDB 3pic)[11], and the glucuronoyl esterase StGE2 of the thermophilic Myceliophthora thermophila (synonym Sporotrichum thermophile, PDB 4g4g, PDB 4g4j)[12]. Consistent with the bacterial MZ0003 having a different substrate specificity to fungal CE15s, significant structural differences were predicted based on sequence alignments[9] This includes the lack of Glu in the conserved position of the catalytic triad, and the presence of three regions that are not found in the fungal enzymes, www.nature.com/scientificreports/. Species Hypocrea jecorina Myceliophtora thermophilia Anabaena variabilis Metagenome Thermus thermophilus Lactobacillus johnsonii Butyrivibrio proteoclasticus Klebsiella pneumoniae Marine sediment metagenome
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