Exploration of three Dyadobacter fermentans enzymes uncovers molecular activity determinants in CE15

Abstract Glucuronoyl esterases (GEs) are serine-type hydrolase enzymes belonging to carbohydrate esterase family 15 (CE15), and they play a central role in the reduction of recalcitrance in plant cell walls by cleaving ester linkages between glucuronoxylan and lignin in lignocellulose. Recent studies have suggested that bacterial CE15 enzymes are more heterogeneous in terms of sequence, structure, and substrate preferences than their fungal counterparts. However, the sequence space of bacterial GEs has still not been fully explored, and further studies on diverse enzymes could provide novel insights into new catalysts of biotechnological interest. To expand our knowledge on this family of enzymes, we investigated three unique CE15 members encoded by Dyadobacter fermentans NS114T, a Gram-negative bacterium found endophytically in maize/corn (Zea mays). The enzymes are dissimilar, sharing ≤ 39% sequence identity to each other‚ and were considerably different in their activities towards synthetic substrates. Combined analysis of their primary sequences and structural predictions aided in establishing hypotheses regarding specificity determinants within CE15, and these were tested using enzyme variants attempting to shift the activity profiles. Together, the results expand our existing knowledge of CE15, shed light into the molecular determinants defining specificity, and support the recent thesis that diverse GEs encoded by a single microorganism may have evolved to fulfil different physiological functions. Key points • D. fermentans encodes three CE15 enzymes with diverse sequences and specificities • The Region 2 inserts in bacterial GEs may directly influence enzyme activity • Rational amino acid substitutions improved the poor activity of the DfCE15A enzyme Supplementary information The online version contains supplementary material available at 10.1007/s00253-024-13175-6.

Table S2.Genomic neighbourhood of DfCE15A.The closest characterized homolog for a neighbouring gene was identified through either primary sequence analysis using BLAST (Altschul et al. 1990) or structural analysis through Foldseek (van Kempen et al. 2023) from the AlphaFold2 (Varadi et al. 2022) predicted structure found on Uniprot (Consortium 2023).DfCE15A is highlighted in green and sequences of a cluster of 3 or more putatively annotated genes associated with a specific function are additionally highlighted.Table S3.Genomic neighbourhood of DfCE15B.The closest characterized homolog for a neighbouring gene was identified through either primary sequence analysis using BLAST (Altschul et al. 1990) S. usitatus,SuCE15A).The residue numbering and secondary structural elements above the alignment are from OtCE15A (PDB: 6GS0).Residues of the catalytic triad and oxyanion stabilizing arginine are shaded in green.Note that, while both bacterial and fungal catalytic histidine residues align structurally, they do not align by primary sequence alignment.Further, fungal CE15 enzyme catalytic acidic residues are found at the position equivalent to residue 290 in OtCE15A, while in bacterial enzymes the acid is found at the position equivalent to residue 356 in OtCE15A, and some bacteria contain both."Region 2", found in bacterial members is highlighted in cyan.Residues targeted for substitution are highlighted in magenta for DfCE15A and orange for DfCE15B.

Fig. S2 :
Fig. S2: Dependency of pH on BnzGlcA hydrolysis catalyzed by DfCE15A (A), DfCE15B (B) and DfCE15C (C).Specific activity with 2 mM BnzGlcA at different pH values was measured in threecomponent buffer as described in the methods.Error bars represent standard errors taken from duplicate measurements with the activity resulting in the highest activity for each enzyme taken as 100%.The rate of ester autohydrolysis increases with increasing pH values, and above pH 9.5 the high rate of autohydrolysis makes accurate determination of the enzymatic cleavage rate unreliable.

Fig. S3 :
Fig. S3: Thermal shift plots of DfCE15s.Assay mixtures contained 5-10 μM of proteins with the temperature increasing by 1 °C/min and changes in fluorescence were quantified relative to a no protein control.

Fig. S4 :
Fig. S4:The overall predicted structures of DfCE15A (A), DfCE15B (B) and DfCE15C (C), coloured relative to the pLDDT confidence values in the colour bar.The predictions are made using AlphaFold(Varadi et al. 2022).Note that all models have a very high confidence score.

Fig. S5 :
Fig. S5: Multiple sequence alignment of the DfCE15 enzymes and selected previously characterized CE15 members.The alignment contains sequences of characterized enzymes from both fungi (C.unicolor, CuGE; and S. thermophile StGE2) and bacteria (O.terrae, OtCE15A; and  S. usitatus, SuCE15A).The residue numbering and secondary structural elements above the alignment are from OtCE15A (PDB: 6GS0).Residues of the catalytic triad and oxyanion stabilizing arginine are shaded in green.Note that, while both bacterial and fungal catalytic histidine residues align structurally, they do not align by primary sequence alignment.Further, fungal CE15 enzyme catalytic acidic residues are found at the position equivalent to residue 290 in OtCE15A, while in bacterial enzymes the acid is found at the position equivalent to residue 356 in OtCE15A, and some bacteria contain both."Region 2", found in bacterial members is highlighted in cyan.Residues targeted for substitution are highlighted in magenta for DfCE15A and orange for DfCE15B.

Fig. S6 :
Fig. S6: Comparison of the overall folds of the DfCE15 protein models.The predicted models of DfCE15A (A), DfCE15B (B), DfCE15C (C) and the experimentally determined model of OtCE15A with the bound glucuronate molecule shown as green sticks (D; PDB accession: 6SYR).The Region 2 in each structure is coloured in cyan.The figure was made using PyMOL 2.5.

Table S4 .
(Consortium 2023)22)023through Foldseek(van Kempen et al. 2023) from the AlphaFold2(Varadi et al. 2022)predicted structure found on Uniprot(Consortium 2023).DfCE15B is highlighted in green and sequences of a cluster of 3 or more putatively annotated genes associated with a specific function are additionally highlighted.Genomic neighbourhood of DfCE15C.The closest characterized homolog for a neighbouring gene was identified through either primary sequence analysis using BLAST(Altschul et al. 1990)or structural analysis through Foldseek(van Kempen et al. 2023) from the AlphaFold2(Varadi et al. 2022)predicted structure found on Uniprot(Consortium 2023).DfCE15C is highlighted in green and sequences of a cluster of 3 or more putatively annotated genes associated with a specific function are additionally highlighted.

Table S5 . Matrix of sequence identity of selected CE15 members.
Representative CE15 members from fungi (fuchsia) and bacteria (blue) shown alongside the members from D. fermentans (green).