Polysaccharide utilization loci from Bacteroidota encode CE15 enzymes with possible roles in cleaving pectin-lignin bonds

Abstract

Lignocellulose is a renewable but complex material exhibiting high recalcitrance to enzymatic hydrolysis, which is attributed, in part, to the presence of covalent linkages between lignin and polysaccharides in the plant cell wall. Glucuronoyl esterases from carbohydrate esterase family 15 (CE15) have been proposed as an aid in reducing this recalcitrance by cleaving ester bonds found between lignin and glucuronoxylan. In the Bacteroidota phylum, some species organize genes related to carbohydrate metabolism in polysaccharide utilization loci (PULs) which encode all necessary proteins to bind, deconstruct, and respond to a target glycan. Bioinformatic analyses identified CE15 members in some PULs that appear to not target the expected glucuronoxylan. Here, five CE15 members from such PULs were investigated with the aim of gaining insights on their biological roles. The selected targets were characterized using glucuronoyl esterase model substrates and with a new synthetic molecule mimicking a putative ester linkage between pectin and lignin. The CE15 enzyme from Phocaeicola vulgatus was structurally determined by X-ray crystallography both with and without carbohydrate ligands with galacturonate binding in a distinct conformation than that of glucuronate. We further explored whether these CE15 enzymes could act akin to pectin methylesterases on pectin-rich biomass but did not find evidence to support the proposed activity. Based on the evidence gathered, the CE15 enzymes in the PULs expected to degrade pectin could be involved in cleavage of uronic acid esters in rhamnogalacturonans.IMPORTANCEThe plant cell wall is a highly complex matrix, and while most of its polymers interact non-covalently, there are also covalent bonds between lignin and carbohydrates. Bonds between xylan and lignin are known, such as the glucuronoyl ester bonds that are cleavable by CE15 enzymes. Our work here indicates that enzymes from CE15 may also have other activities, as we have discovered enzymes in PULs proposed to target other polysaccharides, including pectin. Our study represents the first investigation of such enzymes. Our first hypothesis that the enzymes would act as pectin methylesterases was shown to be false, and we instead propose that they may cleave other esters on complex pectins such as rhamnogalacturonan II. The work presents both the characterization of five novel enzymes and can also provide indirect information about the components of the cell wall itself, which is a highly challenging material to chemically analyze in fine detail.

Keywords: carbohydrate esterase; carbohydrate esterase family 15; glucuronoyl esterase; lignocellulose; pectin; protein structure.

Fig 1

Structures of the model substrates used in the study. From left to right: methyl glucuronate, allyl glucuronate, benzyl-glucuronate, methyl galacturonate, and benzyl galacturonate.

Fig 2

Overview of the organization of the PULs encoding the investigated CE15 enzymes, as predicted by the PULDB (35). Encoding species are listed above each PUL (with former names in parentheses where applicable), and locus tags are above each corresponding gene, drawn to scale. The top two PULs appear to target pectin; the middle might possibly target pectin-derived oligosaccharides, and the bottom two are unlikely to target pectin. Functional annotations are shown inside each gene symbol and are color coded following the PULDB color scheme: glycoside hydrolases in pink, proteins of unknown function in light gray, SusD-like binding proteins in orange, SusC-like transport proteins in violet, regulatory hybrid two-component systems and other regulatory factors (HTCSs, σ) in light blue, carbohydrate esterases in brown, polysaccharide lyases in purple, and carbohydrate-binding modules in green.

Fig 3

Effect of PvCE15 on glucose release during a 1-h saccharification of certain pectin-rich biomasses. Biomass materials of (A) carrot pomace, (B) potato peels, and (C) orange peels were milled, washed, and resuspended in assays of 5 mg/mL and incubated for 1 h at 30°C with either UltraFlo (UF), PvCE15 (CE15), pectate lyase (PL), or pectin methyl esterase (PME) and were compared to control reactions (C) lacking the addition of enzymes. Released monosaccharides from assays (N = 4) were quantified by ion chromatography, and the differences in means were evaluated by Student’s t-test. The addition of PvCE15 to either UltraFlo or UltraFlo supplemented with the pectate lyase did not lead to significant increases (ns) in released glucose, while the addition of PME to the reactions with UltraFlo supplemented with the pectate lyase did for both carrot pomace and potato peels but not orange peels. The washed orange peel control samples contained a considerable amount of free glucose that was not present before the 1-h incubation, indicating additional factors were contributing to monosaccharide release during the assays.

Fig 4

Overall structure of PvCE15. A cartoon representation of PvCE15 (left, PDB accession: 8Q6S) in complex with GlcA, shown in yellow sticks, with the residues of the catalytic triad shown as green sticks and the regions corresponding to Reg2 and RegN shown in cyan and orange, respectively. A surface representation (right) illustrating the buildup of the binding cleft by Reg2 and RegN.

Fig 5

Active site organization of PvCE15 in complex with uronate substrates. The active site of PvCE15 (A, PDB accession: 8Q6S) in complex with glucuronate (B, PDB accession: 8QCL), and galacturonate (C, PDB accession: 8QEF) compared to the active site of OtCE15A (D, PDB accession: 6GS0) in complex with glucuronate (E, PDB accession: 6SYR) and galacturonate (F, PDB accession: 6SZO). Residues of the catalytic triad and the additional catalytic acidic residue in OtCE15A are highlighted in green, and the water and DMSO molecules found in the galacturonate complex structures are highlighted in red and yellow, respectively. The galacturonate complex with OtCE15A was obtained with the catalytic serine substitution (S267A). Key interaction distances ≤3 Å are shown as black dashes.