Structural insights into glycoside hydrolase family 32 and 68 enzymes: functional implications

Abstract

Glycoside hydrolases (GH) have been shown to play unique roles in various biological processes like the biosynthesis of glycans, cell wall metabolism, plant defence, signalling, and the mobilization of storage reserves. To date, GH are divided into more than 100 families based upon their overall structure. GH32 and GH68 are combined in clan GH-J, not only harbouring typical hydrolases but also non-Leloir type transferases (fructosyltransferases), involved in fructan biosynthesis. This review summarizes the recent structure-function research progress on plant GH32 enzymes, and highlights the similarities and differences compared with the microbial GH32 and GH68 enzymes. A profound analysis of ligand-bound structures and site-directed mutagenesis experiments identified key residues in substrate (or inhibitor) binding and recognition. In particular, sucrose can bind as inhibitor in Cichorium intybus 1-FEH IIa, whereas it binds as substrate in Bacillus subtilis levansucrase and Arabidopsis thaliana cell wall invertase (AtcwINV1). In plant GH32, a single residue, the equivalent of Asp239 in AtcwINV1, appears to be important for sucrose stabilization in the active site and essential in determining sucrose donor specificity.

Understanding the Role of Defective Invertases in Plants: Tobacco Nin88 Fails to Degrade Sucrose

“…Neutral/alkaline invertases (A/N-Invs) are nonglycosylated proteins characterized by a neutral to alkaline pH optimum classified in glycoside hydrolase family 100 (GH100; Sturm et al, 1999;Lammens et al, 2009). These enzymes have long remained poorly studied, and for decades, A/N-Invs were believed to occur exclusively in the cytosol.…”

“…Acid invertases are glycosylated proteins localized in the vacuole (vacuole invertases [vINVs]) or in the apoplast (cell wall invertases [cwINVs]) and belong to the family GH32, together with plant fructan-metabolizing enzymes. Close relationships between vINVs and fructan-biosynthesizing enzymes, on the one hand, and cwINVs and fructanbreakdown enzymes, on the other hand, were described (Ritsema et al, 2006;Lammens et al, 2009). vINVs play important roles in cell expansion (Wang et al, 2010), and they determine the sugar composition in sugar-storing sink organs (Tang et al, 1999;Bhaskar et al, 2010).…”

Crystal Structures of Aspergillus japonicus Fructosyltransferase Complex with Donor/Acceptor Substrates Reveal Complete Subsites in the Active Site for Catalysis

“…Enzymes of both families GH32 and GH68 comprise clan GH-J according to their folding similarities. In this clan there are six three-dimensional structures available, including four enzymes from microbial origins and two from plants (17): levansucrase and its mutants from Bacillus subtilis (PDB: 1OYG) (18,19), levansucrase from Gluconacetobacter diazotrophicus (1W18) (20), invertase from Thermotoga maritima (1UYP and 1W2T) (21,22), cell-wall invertase (2AC1) (23) and its mutant complexes with sucrose from Arabidopsis thaliana (2QQW, 2QQU, and 2QQV) (24), exoinulinase from Aspergillus awamori (1W4Y and 1Y9G) (25), and fructan 1-exohydrolase IIa (1-FEH IIa) from Cichorium intybus and its mutant complexes with various substrates (1ST8, 2ADD, and 2AEZ) (26,27).…”

Structural Modelling and Structure-Function Analysis of <i>Zymomonas mobilis</i> Levansucrase

“…Levansucrases of Gram-positive bacteria (such as B. subtilis, Bacillus megaterium and Streptococcus salivarius) produce generally levan without accumulating the intermediate FOS [14,15,16], while levansucrases from some Gram-negative bacteria (such as G. diazotrophicus, E. amylovora and Pseudomonas syringae) produce high amounts of FOS with small amounts of levan [17,18,19]. Structural studies recently pointed out that the main differences between FOS and levan-producing enzymes are located on the surface loops [12,20]. Levansucrase from Z. mobilis; on the other hand, can produce both levan and FOS depending on the reaction conditions [9,21,22,23].…”