Structure of a GH51 α-L-arabinofuranosidase from Meripilus giganteus: conserved substrate recognition from bacteria to fungi

Abstract

α-L-Arabinofuranosidases from glycoside hydrolase family 51 use a stereochemically retaining hydrolytic mechanism to liberate nonreducing terminal α-L-arabinofuranose residues from plant polysaccharides such as arabinoxylan and arabinan. To date, more than ten fungal GH51 α-L-arabinofuranosidases have been functionally characterized, yet no structure of a fungal GH51 enzyme has been solved. In contrast, seven bacterial GH51 enzyme structures, with low sequence similarity to the fungal GH51 enzymes, have been determined. Here, the crystallization and structural characterization of MgGH51, an industrially relevant GH51 α-L-arabinofuranosidase cloned from Meripilus giganteus, are reported. Three crystal forms were grown in different crystallization conditions. The unliganded structure was solved using sulfur SAD data collected from a single crystal using the I23 in vacuo diffraction beamline at Diamond Light Source. Crystal soaks with arabinose, 1,4-dideoxy-1,4-imino-L-arabinitol and two cyclophellitol-derived arabinose mimics reveal a conserved catalytic site and conformational itinerary between fungal and bacterial GH51 α-L-arabinofuranosidases.

The In Silico Characterization of Monocotyledonous α-l-Arabinofuranosidases on the Example of Maize

“…A homologous N-terminal domain was found in the MgGH51 enzyme of the fungus M. giganteus . It forms a ten-strand β-sandwich structure similar to the bacterial CBM4 domains [ 31 ] ( Figure 5 A,B, highlighted in purple). However, unlike the bacterial CBM4s that bind cellulose, laminarin, and xylan oligosaccharides [ 72 , 73 , 74 , 75 ], the N-terminal domain of MgGH51 was unable to bind saccharides and was named a CBM4-like domain [ 31 ].…”

“…The beginning and the end of the CBM4-like domain were established by L-x-V-D and S-x-x-P conserved motifs, respectively ( Figure S1 ). The S-x-x-P motif is located on the loop separating the CBM4-like domain from the others in the MgGH51 structure [ 31 ]. The generated CBM4-like domain profile was represented in 119 of 133 (89.5%) GH51 plant sequences ( Table S1 ) that were used for phylogenetic analysis earlier ( Figure 3 ).…”

“…In contrast, the G-x-x-F-E-x-I-x-x-x-G-x-G-G motif which is conserved in CBM4-like domains ( Figure S1 , black frame) is absent or significantly altered in bacterial CBM4s ( Figure S3 , black frame). This motif is located in the β-strand that permeates the entire spatial structure of the MgGH51 enzyme and forms the bottom of its catalytic pocket ( Figure 5 A,B, highlighted in blue) [ 31 ].…”

“…Typical enzymes also possess a C-terminal β-sandwich domain that stabilizes the overall structure [ 25 , 26 , 27 , 28 , 29 , 30 ]. However, a recent study of the fungal enzyme MgGH51 showed the presence of an N-terminal domain similar to the carbohydrate-binding module (CBM) of family 4 with unknown function in addition to the aforementioned domains [ 31 ].…”

The chemistry and biology of natural ribomimetics and related compounds

“…11). 64,65 Some plants and bacteria also produce 2,5-dideoxy-2,5imino-D-mannitol (DMDP, 58) and related compounds, e.g., 6deoxy-DMAP (2,5-imino-1,2,5-trideoxy-D-mannitol, 59), 66 homo-DMAP (2,5-dideoxy-2,5-imino-DL-glycero-D-manno-heptitol, 60), 67 3,6-deoxy-homo-DMDP (61), 68,69 and 2,5-imino-2,5,6-trideoxy-Dgulo-heptitol (62) (Fig. 12).…”