anaerobic digester metagenome CE1 of multi-enzyme CE1-GH62-GH10 wtsFae1A
Comment
mutidomain enzyme consisting of three catalytic domains: ferulic acid esterase, arabinofuranosidase, and xylanase CE1-GH62-GH10. Here only N-terminal carbohydrate binding module family 48 and ferulic acid esterase (CE1) from the multi-enzyme CE1-GH62-GH10
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > unclassified sequences: NE > metagenomes: NE > ecological metagenomes: NE > anaerobic digester metagenome: NE
No mutation 1 structure: 6RZO: Crystal structure of the N-terminal carbohydrate binding module family 48 and ferulic acid esterase from the multi-enzyme CE1-GH62-GH10 wtsFae1A 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 MEDFKPTSTNQPGRQYPQVNSEGRVRARIEAPQAHTVLLDIGGVRYPMTQ GEDGAWIGDSRPQDEGFHYYQLVIDGARVPDPGSLYFFGANRWGSGVEVP AHDQDFYAIKDVPHGRVQKILFPSGSTSTIRRAFVYTPPDYGKDLSKRYP VLYLQHGWGEDETGWANQGRVNLIMDNLIAEGKARPFIIVMTYGMTNEIR FGGIREFDIRPFQTVLVDELIPYIDANFRTRSDQPHRAMAGLSMGGMETR LITMNNLDLFSHIGLFSGGTISASDITDRDVFKQKIKLVFVSCGSRENPG RFRPAVDSLQQAGISAVSYVSPDTAHEWQTWRRSFYQFAQLLFQ
Feruloyl esterases (FAEs, EC 3.1.1.73) catalyze the hydrolytic cleavage of ester bonds between feruloyl and arabinosyl moieties in arabinoxylans. Recently, we discovered that two bacterial FAEs could catalyze release of diferulic acid moieties (diFAs) from highly substituted, cross-linked corn bran arabinoxylan. Here, we show that several fungal FAEs, notably AnFae1 (Aspergillus niger), AoFae1 (A. oryzae), and MgFae1 (Magnaporthe oryzae (also known as M. grisae)) also catalyze liberation of diFAs from complex arabinoxylan. By comparing the enzyme kinetics of diFA release to feruloyl esterase activity of the enzymes on methyl- and arabinosyl-ferulate substrates we demonstrate that the diFA release activity cannot be predicted from the activity of the enzymes on these synthetic substrates. A detailed structure-function analysis, based on AlphaFold2 modeled enzyme structures and docking with the relevant di-feruloyl ligands, reveal how distinct differences in the active site topology and surroundings may explain the diFA releasing action of the enzymes. Interestingly, the analysis also unveils that the carbohydrate binding module of the MgFae1 may play a key role in the diFA releasing ability of this enzyme. The findings contribute further understanding of the function of FAEs in the deconstruction of complex arabinoxylans and provide new opportunities for enzyme assisted upgrading of complex bran arabinoxylans.
A three catalytic domain multi-enzyme; a CE1 ferulic acid esterase, a GH62 alpha-l-arabinofuranosidase and a GH10 beta-d-1,4-xylanase was identified in a metagenome obtained from wastewater treatment sludge. The capability of the CE1-GH62-GH10 multi-enzyme to degrade arabinoxylan was investigated to examine the hypothesis that CE1-GH62-GH10 would degrade arabinoxylan more efficiently than the corresponding equimolar mix of the individual enzymes. CE1-GH62-GH10 efficiently catalyzed the production of xylopyranose, xylobiose, xylotriose, arabinofuranose and ferulic acid (FA) when incubated with insoluble wheat arabinoxylan (WAX-I) (kcat = 20.8 +/- 2.6 s(-1)). Surprisingly, in an equimolar mix of the individual enzymes a similar kcat towards WAX-I was observed (kcat = 17.3 +/- 3.8 s(-1)). Similarly, when assayed on complex plant biomass the activity was comparable between CE1-GH62-GH10 and an equimolar mix of the individual enzymes. This suggests that from a hydrolytic point of view a CE1-GH62-GH10 multi-enzyme is not an advantage. Determination of the melting temperatures for CE1-GH62-GH10 (71.0 +/- 0.05 degrees C) and CE1 (69.9 +/- 0.02), GH62 (65.7 +/- 0.06) and GH10 (71 +/- 0.05 degrees C) indicates that CE1 and GH62 are less stable as single domain enzymes. This conclusion was corroborated by the findings that CE1 lost 50% activity within 2 h, while GH62 retained 50% activity after 24 h, whereas CE1-GH62-GH10 and GH10 retained 50% activity for 72 h. GH62-GH10, when appended to each other, displayed a higher specificity constant (kcat/Km = 0.3 s(-1) mg(-1) ml) than the individual GH10 (kcat/Km = 0.12 s(-1) +/- 0.02 mg(-1) ml) indicating a synergistic action between the two. Surprisingly, CE1-GH62, displayed a 2-fold lower kcat towards WAX-I than GH62, which might be due to the presence of a putative carbohydrate binding module appended to CE1 at the N-terminal. Both CE1 and CE1-GH62 released insignificant amounts of FA from WAX-I, but FA was released from WAX-I when both CE1 and GH10 were present, which might be due to GH10 releasing soluble oligosaccharides that CE1 can utilize as substrate. CE1 also displayed activity towards solubilized 5-O-trans-feruloyl-alpha-l-Araf (kcat = 36.35 s(-1)). This suggests that CE1 preferably acts on soluble oligosaccharides.
Feruloyl esterases (EC 3.1.1.73), belonging to carbohydrate esterase family 1 (CE1), hydrolyze ester bonds between ferulic acid (FA) and arabinose moieties in arabinoxylans. Recently, some CE1 enzymes identified in metagenomics studies have been predicted to contain a family 48 carbohydrate-binding module (CBM48), a CBM family associated with starch binding. Two of these CE1s, wastewater treatment sludge (wts) Fae1A and wtsFae1B isolated from wastewater treatment surplus sludge, have a cognate CBM48 domain and are feruloyl esterases, and wtsFae1A binds arabinoxylan. Here, we show that wtsFae1B also binds to arabinoxylan and that neither binds starch. Surface plasmon resonance analysis revealed that wtsFae1B's Kd for xylohexaose is 14.8 mum and that it does not bind to starch mimics, beta-cyclodextrin, or maltohexaose. Interestingly, in the absence of CBM48 domains, the CE1 regions from wtsFae1A and wtsFae1B did not bind arabinoxylan and were also unable to catalyze FA release from arabinoxylan. Pretreatment with a beta-d-1,4-xylanase did enable CE1 domain-mediated FA release from arabinoxylan in the absence of CBM48, indicating that CBM48 is essential for the CE1 activity on the polysaccharide. Crystal structures of wtsFae1A (at 1.63 A resolution) and wtsFae1B (1.98 A) revealed that both are folded proteins comprising structurally-conserved hydrogen bonds that lock the CBM48 position relative to that of the CE1 domain. wtsFae1A docking indicated that both enzymes accommodate the arabinoxylan backbone in a cleft at the CE1-CBM48 domain interface. Binding at this cleft appears to enable CE1 activities on polymeric arabinoxylan, illustrating an unexpected and crucial role of CBM48 domains for accommodating arabinoxylan.
anaerobic digester metagenome CE1 of multi-enzyme CE1-GH62-GH10 wtsFae1A
