Abstract
A saprophytic Bacillus subtilis secretes two types of rhamnogalacturonan (RG) lyases, endotype YesW and exotype YesX, which are responsible for an initial cleavage of the RG type I (RG-I) region of plant cell wall pectin. Polysaccharide lyase family 11 YesW and YesX with a significant sequence identity (67.8%) cleave glycoside bonds between rhamnose and galacturonic acid residues in RG-I through a beta-elimination reaction. Here we show the structural determinants for substrate recognition and the mode of action in polysaccharide lyase family 11 lyases. The crystal structures of YesW in complex with rhamnose and ligand-free YesX were determined at 1.32 and 1.65 A resolution, respectively. The YesW amino acid residues such as Asn(152), Asp(172), Asn(532), Gly(533), Thr(534), and Tyr(595) in the active cleft bind to rhamnose molecules through hydrogen bonds and van der Waals contacts. Other rhamnose molecules are accommodated at the noncatalytic domain far from the active cleft, revealing that the domain possibly functions as a novel carbohydrate-binding module. A structural comparison between YesW and YesX indicates that a specific loop in YesX for recognizing the terminal saccharide molecule sterically inhibits penetration of the polymer over the active cleft. The loop-deficient YesX mutant exhibits YesW-like endotype activity, demonstrating that molecular conversion regarding the mode of action is achieved by the addition/removal of the loop for recognizing the terminal saccharide. This is the first report on a structural insight into RG-I recognition and molecular conversion of exotype to endotype in polysaccharide lyases.
Highlights
Lies based on their amino acid sequences in the CarbohydrateActive enZymes (CAZy) data base (1)
Little knowledge has been accumulated on the mechanisms of substrate recognition and catalytic reaction in lyases acting on the rhamnogalacturonan (RG) region of pectin
Polygalacturonan is present as a linear backbone, and RG type I (RG-I) and RG type II (RG-II) are attached to the backbone as branched chains (11–13)
Summary
Strain A1, three endotype alginate lyases, A1-I, A1-II, and A1-III, release oligosaccharides from the polymer, and the resultant oligoalginates are converted to the constituent monosaccharides through the reaction of exotype lyase, A1-IV (19). Structure and function relationships in the endotype PLs have been demonstrated (2, 3), but little information on the structural features of exotype lyases has been accumulated. Endotype YesW and exotype YesX from B. subtilis significantly resemble each other in primary structure, i.e. 68.7% identity in 597-amino acid overlap (16), suggesting that their mode of action (endo/exo) is determined by a slight structural difference present in the catalytic domain. On the basis of this structure and function relationship, exotype YesX was converted into YesW-like endotype enzyme by protein engineering
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