Rhamnogalacturonan lyase reveals a unique three-domain modular structure for polysaccharide lyase family 4*1

Abstract

Rhamnogalacturonan lyase (RG-lyase) specifically recognizes and cleaves a-1,4 glycosidic bonds between L L -rhamnose and D D -galacturonic acids in the backbone of rhamno galacturonan-I, a major component of the plant cell wall polysaccharide, pectin. The three-dimensional structure of RGlyase from Aspergillus aculeatus has been determined to 1.5 A resolution representing the first known structure from polysaccharide lyase family 4 and of an enzyme with this catalytic specificity. The 508-amino acid polypeptide displays a unique arrangement of three distinct modular domains. Each domain shows structural homology to non-catalytic domains from other carbohydrate active enzymes.

Analysis of carbohydrate binding in a family 5 exoglucanase

“…The catalytically disabled mutant E292Q did not show coherent electron density to indicate stable binding of oligosaccharide substrates but unexpectedly revealed an external glucose binding site that may provide a foothold for the yeast cell wall Dglucan. Exg was converted to a glucosynthase via E292S and crystallized in the presence of donor 1-fluoro-α-Dglucose and acceptor p-nitrophenyl-ß-D-glucopyranoside [2]. The two sugars were oriented such that donor sugar C1 was close to acceptor O6, consistent with nmr analysis of solution products which showed the main product to be ß-1,6-linked disaccharide and not ß-1,3-as expected.…”

“…Rhamnogalacturonan lyase (AA-RGL) from A. aculeatus is a pectin backbone degrading enzyme that belongs to the polysaccharide lyase family 4 [1]. The structure of AA-RGL [2] was previously determined at a resolution of 1.5 Å. The enzyme is comprised of three domains.…”

“…The domains display structural homology with domains from other carbohydrate active enzymes, but neither of these domains possess any catalytic activity. Based on sequence alignments within polysaccharide lyase family 4 the location of the active site was proposed and possible candidates for the catalytic base and other active residues were identified [2]. Two of these putative active site residues were mutated (K150A and H210A).…”

Crystal Structure of Exotype Alginate Lyase Atu3025 from Agrobacterium tumefaciens

“…The crystal structures of PLs in 18 families have been determined, and the structure and functional relationships of enzymes such as pectin lyase in family PL-1 (14); pectate lyases in families PL-2, PL-3, and PL-10 (15-17); alginate lyases in families PL-5, PL-7, and PL-14 (13,18,19); rhamnogalacturonan lyases in families PL-4 and PL-11 (20,21); chondroitin lyases in families PL-6 and PL-8 (22); hyaluronan lyases in families PL-8 and PL-16 (23,24); heparin lyases in families PL-13 and PL-21 (25,26); and xanthan lyase in family PL-8 (12) have been demonstrated. On the other hand, no structural information on family PL-15 alginate lyases has been accumulated, and the enzyme mechanism for releasing unsaturated monosaccharides from the polysaccharide main chain remains to be clarified.…”

A Novel Structural Fold in Polysaccharide Lyases

“…The crystal structure of a family PL-4 RG lyase, RhgB, has been determined from Aspergillus aculeatus KSM 510 (33), although its structure and function relationship remains to be clarified. Family PL-11 RG lyases Rgl11A from Cellvibrio japonicus (34), Rgl11Y from Clostridium cellulolyticum (35), and YesW and YesX from B. subtilis (24) have been characterized enzymatically, but no structural analysis of these enzymes has, to our knowledge, been reported.…”