(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > PVC group: NE > Verrucomicrobia: NE > Opitutae: NE > Opitutales: NE > Opitutaceae: NE > Opitutus: NE > Opitutus terrae: NE
No mutation 11 structures(e.g. : 6GRW, 6GS0, 6SYR... more)(less) 6GRW: Glucuronoyl Esterase from Opitutus terrae OtCE15A (Au derivative), 6GS0: Glucuronoyl Esterase from Opitutus terrae OtCE15A native, 6SYR: Glucuronoyl Esterase from Opitutus terrae OtCE15A-Wt-GlcA, 6SYU: The wild type glucuronoyl esterase OtCE15A from Opitutus terrae in complex with xylobiose, 6SYV: Glucuronoyl Esterase from Opitutus terrae OtCE15A-S267A-GlcA, 6SZ0: The glucuronoyl esterase OtCE15A H408A variant from Opitutus terrae, 6SZ4: The glucuronoyl esterase OtCE15A H408A variant from Opitutus terrae in complex with, and covalently linked to, D-glucuronate, 6SZO: Glucuronoyl Esterase from Opitutus terrae OtCE15A-S267A-GalA, 6T0E: The glucuronoyl esterase OtCE15A S267A variant from Opitutus terrae in complex with benzyl D-glucuronoate and D-glucuronate, 6T0I: The wild type glucuronoyl esterase OtCE15A from Opitutus terrae in complex with the aldotetrauronic acid XUX, 7B7H: The glucuronoyl esterase OtCE15A R268A variant from Opitutus terrae in complex with, and covalently linked to, D-glucuronate No kinetic
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MRNVLAALSLLFTLTSMQATSRPARLDDTPPPAYTLPDPLVGADGTRVHD RATWQHRRRPELLQLFAREVYGRTPLGRPEGMVFKVTTMEHAALGGAATR KEVTVRFGRDPNAPSMQLLLYVPNAVIARAERAPVFLGLNFYGNHTVHTD PAIALSARWIPAEAPNGANHRATEAARGSDAQKWPVEQILARGYAVATVY CGDLCPDRPDGLNASVASWLDAAAGDQRAPDAWGAIGVWAWGLSRALDYL ETDPLVDASRVAVHGHSRLGKAALWAGAQDDRFALVISNESGCGGAALSK RIHGETVARINTVFPHWFARNFRRYDDHEEALPVDQHELLALVAPRPLYV ASAEDDDWADPRGEFLAVKAAEPVFRLFGQTGPSGEDVPRVNEPSGGALR YHIRPGPHGMTAQDWAFYLAFADEWLKSALPAREPQR
Glucuronoyl esterases (GEs) are alpha/beta serine hydrolases and a relatively new addition in the toolbox to reduce the recalcitrance of lignocellulose, the biggest obstacle in cost-effective utilization of this important renewable resource. While biochemical and structural characterization of GEs have progressed greatly recently, there have yet been no mechanistic studies shedding light onto the rate-limiting steps relevant for biomass conversion. The bacterial GE OtCE15A possesses a classical yet distinctive catalytic machinery, with easily identifiable catalytic Ser/His completed by two acidic residues (Glu and Asp) rather than one as in the classical triad, and an Arg side chain participating in the oxyanion hole. By QM/MM calculations, we identified deacylation as the decisive step in catalysis, and quantified the role of Asp, Glu and Arg, showing the latter to be particularly important. The results agree well with experimental and structural data. We further calculated the free-energy barrier of post-catalysis dissociation from a complex natural substrate, suggesting that in industrial settings non-catalytic processes may constitute the rate-limiting step, and pointing to future directions for enzyme engineering in biomass utilization.
Title: Structural and biochemical studies of the glucuronoyl esterase OtCE15A illuminate its interaction with lignocellulosic components Mazurkewich S, Poulsen JN, Lo Leggio L, Larsbrink J Ref: Journal of Biological Chemistry, 294:19978, 2019 : PubMed
Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages between lignin and glucuronic acid moieties on glucuronoxylan in plant biomass. As such, GEs represent promising biochemical tools in industrial processing of these chemically recalcitrant materials. However, details on how GEs interact and catalyze degradation of their natural substrates are sparse, calling for thorough enzyme structure-function studies. GEs belong to carbohydrate esterase family 15 (CE15), which is part of the larger alpha/beta hydrolase superfamily. We present here a structural and mechanistic investigation of the bacterial GE OtCE15A. GEs contain a Ser-His-Asp/Glu catalytic triad, but the location of the catalytic acid in GEs is known to be variable, and OtCE15A possesses two putative catalytic acidic residues in its active site. Through site-directed mutagenesis, we demonstrate here that these residues are functionally redundant, possibly indicating the evolutionary route toward new functionalities within the CE15 family. Structures determined with the bound products glucuronate and galacturonate, as well as a covalently bound intermediate, provided insights into the catalytic mechanism of CE15. A structure of OtCE15A with the glucuronoxylooligosaccharide 2(3)-(4-O-methyl-alpha-D-glucuronyl)-xylotriose (XUX) disclosed that the enzyme can indeed interact with polysaccharides from the plant cell wall, and an additional structure with the disaccharide xylobiose revealed an enzyme surface binding site that might indicate a mechanism by which the enzyme recognizes long glucuronoxylan chains. These results indicate that OtCE15A, and likely most CE15 family enzymes, can utilize glucuronoxylooligosaccharide esters and support the proposal that these enzymes are active on lignin-carbohydrate complexes in plant biomass.
Background: Lignocellulose 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. Results: Ten 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. Conclusions: Bacterial 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.
Opitutus terrae (strain DSM 11246 / PB90-1) OtCE15A Glucuronoyl Esterase
