BackgroundLignocellulose is highly recalcitrant to enzymatic deconstruction, where the recalcitrance primarily results from chemical linkages between lignin and carbohydrates. Glucuronoyl esterases (GEs) from carbohydrate esterase family 15 (CE15) have been suggested to play key roles in reducing lignocellulose recalcitrance by cleaving covalent ester bonds found between lignin and glucuronoxylan. However, only a limited number of GEs have been biochemically characterized and structurally determined to date, limiting our understanding of these enzymes and their potential exploration.ResultsTen CE15 enzymes from three bacterial species, sharing as little as 20% sequence identity, were characterized on a range of model substrates; two protein structures were solved, and insights into their regulation and biological roles were gained through gene expression analysis and enzymatic assays on complex biomass. Several enzymes with higher catalytic efficiencies on a wider range of model substrates than previously characterized fungal GEs were identified. Similarities and differences regarding substrate specificity between the investigated GEs were observed and putatively linked to their positioning in the CE15 phylogenetic tree. The bacterial GEs were able to utilize substrates lacking 4-OH methyl substitutions, known to be important for fungal enzymes. In addition, certain bacterial GEs were able to efficiently cleave esters of galacturonate, a functionality not previously described within the family. The two solved structures revealed similar overall folds to known structures, but also indicated active site regions allowing for more promiscuous substrate specificities. The gene expression analysis demonstrated that bacterial GE-encoding genes were differentially expressed as response to different carbon sources. Further, improved enzymatic saccharification of milled corn cob by a commercial lignocellulolytic enzyme cocktail when supplemented with GEs showcased their synergistic potential with other enzyme types on native biomass.ConclusionsBacterial GEs exhibit much larger diversity than fungal counterparts. In this study, we significantly expanded the existing knowledge on CE15 with the in-depth characterization of ten bacterial GEs broadly spanning the phylogenetic tree, and also presented two novel enzyme structures. Variations in transcriptional responses of CE15-encoding genes under different growth conditions suggest nonredundant functions for enzymes found in species with multiple CE15 genes and further illuminate the importance of GEs in native lignin–carbohydrate disassembly.Electronic supplementary materialThe online version of this article (10.1186/s13068-018-1213-x) contains supplementary material, which is available to authorized users.

4-Hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-oxoadipate (HMG/CHA) aldolase from Pseudomonas putida F1 catalyzes the last step of the bacterial protocatechuate 4,5-cleavage pathway. The preferred substrates of the enzyme are 2-keto-4-hydroxy acids with a 4-carboxylate substitution. The enzyme also exhibits oxaloacetate decarboxylation and pyruvate ␣-proton exchange activity. Sodium oxalate is a competitive inhibitor of the aldolase reaction. The pH dependence of k cat /K m and k cat for the enzyme is consistent with a single deprotonation with pK a values of 8.0 ؎ 0.1 and 7.0 ؎ 0.1 for free enzyme and enzyme substrate complex, respectively. The 1.8 Å x-ray structure shows a four-layered ␣-␤-␤-␣ sandwich structure with the active site at the interface of two adjacent subunits of a hexamer; this fold resembles the RNase E inhibitor, RraA, but is novel for an aldolase. The catalytic site contains a magnesium ion ligated by Asp-124 as well as three water molecules bound by Asp-102 and Glu-199. A pyruvate molecule binds the magnesium ion through both carboxylate and keto oxygen atoms, completing the octahedral geometry. The carbonyl oxygen also forms hydrogen bonds with the guanadinium group of Arg-123, which site-directed mutagenesis confirms is essential for catalysis. A mechanism for HMG/CHA aldolase is proposed on the basis of the structure, kinetics, and previously established features of other aldolase mechanisms.

Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages found between lignin and glucuronic acid moieties on glucuronoxylan in plant biomass. As such, GEs represent promising biochemical tools in industrial processing of these recalcitrant resources. However, details on how GEs interact with their natural substrates are sparse, calling for thorough structure-function studies. Presented here is the structure and biochemical characterization of a GE, Tt CE15A, from the bacterium Teredinibacter turnerae , a symbiont of wood-boring shipworms. To gain deeper insight into enzyme–substrate interactions, inhibition studies were performed with both the WT Tt CE15A and variants in which we, by using site-directed mutagenesis, substituted residues suggested to have key roles in binding to or interacting with the aromatic and carbohydrate structures of its uronic acid ester substrates. Our results support the hypothesis that two aromatic residues (Phe-174 and Trp-376), conserved in bacterial GEs, interact with aromatic and carbohydrate structures of these substrates in the enzyme active site, respectively. The solved crystal structure of Tt CE15A revealed features previously not observed in either fungal or bacterial GEs, with a large inserted N-terminal region neighboring the active site and a differently positioned residue of the catalytic triad. The findings highlight key interactions between GEs and complex lignin-carbohydrate ester substrates and advance our understanding of the substrate specificities of these enzymes in biomass conversion.