Structural basis for xyloglucan specificity within GH5 family and the molecular determinants for a-D-Xylp(1¿6)-D-Glcp recognition at the -1 subsite

Authors: SANTOS, C - Brazilian Biosciences National Laboratory (LNBIO); CORDEIRO, R - Brazilian Biosciences National Laboratory (LNBIO); Wong, Dominic; MURAKAMI, M - Brazilian Biosciences National Laboratory (LNBIO)

Submitted to: Biochemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/23/2015
Publication Date: 2/25/2015
Citation: Santos, C.R., Cordeiro, R.L., Wong, D., Murakami, M.T. 2015. Structural basis for xyloglucan specificity within GH5 family and the molecular determinants for a-D-Xylp(1¿6)-D-Glcp recognition at the -1 subsite. Biochemistry. 54:1930-1942.

Interpretive Summary: Plant cell walls of many nongraminaceous species contain xyloglucans as the most abundant hemicellulose. Xyloglucans are often crosslinked to cellulose fibers, and they are an important target for fungal enzymes to achieve complete degradation of the cell wall. Two types of enzymes, endo- and exo-xyloglucanases have been extensively studied in our previous work. The genes were isolated by screening of a rumen microbial metagenomic, cloned and expressed in E. coli, and the enzymes purified and characterized. In the present study, the crystal structures of the two enzymes were solved in the native state and in complex with xyloglucan oligosaccharides. The substrate-binding pattern and mechanism were examined with great details for the first time, yielding subsite maps at high resolution. These structural revelations would lead to improving degradation of xyloglucan polymers, with biotechnological implications, such as xyloglucan gels for drug delivery and cellulosic fiber modification.

Technical Abstract: GH5 is one of the most versatile and largest glycoside hydrolase families, comprising at least 20 distinct activities within a common structural scaffold. However, the molecular basis for the functional differentiation among GH5 members is still not fully understood, principally for xyloglucan specificity. In this work, we elucidated the high-resolution crystal structures of two novel GH5 xyloglucanases retrieved from a rumen microflora metagenomic library, in the native state and in complex with xyloglucan-derived oligosaccharides. These results provided insights into the structural determinants that differentiate GH5 xyloglucanases from parental cellulases and also a new mode of action within the GH5 family related to the -1 subsite topology. The oligosaccharide found in the aglycone region of XEG5A complex, permitted to map, for the first time, the positive subsites of a GH5 xyloglucanase, revealing the importance of a structural adaptation in the +1 subsite to confer the ability of some GH5 enzymes to attack xyloglucan. Complementarily, the XEG5B complex covered the glycone region completing the subsite mapping of GH5 xyloglucanases at high resolution. Interestingly, XEG5B is, to date, the only GH5 member able to cleave XXXG into XX and XG, and in the light of these results, we propose that a modification in the -1 subsite enables the accommodation of a xylosyl group at this position. The stereo-chemical compatibility with ramification at the -1 subsite was also reported for a structurally non-related xyloglucanase belonging to the GH74 family, indicating to be an essential attribute for this mode of action.