Plant chitinases use two different hydrolytic mechanisms

Abstract

Bacterial, fungalG animal, and some plant ehNnases form fmily 18 of glycosyl hydrelases. Most plant ehitinases form the family 19, While sense ¢kitinases also have lysozyme activity, animal ~mzymes belong to different families. For 81ycesyl hydrobmes, two reaction mechanisms are possible, lendin8 to either retention or inversion of the anomerlc conflauratlon. We analyzed by HPLC the stereoehemleal ontcome of the hydrolysis catalyzed by cucumber and bean chitlnaes~ belonging to families 18 and 19, respectively. Cucumber ehltlnue used the reteJnlng mechanism as known for bacterial cldtlmmeJ and hen eag white lysozyme for which the mechanlmn hun been determined. In contrast, bean ¢ltltinase utalyzed the hydrolysis of cldtoollgosaeehafldes with overall inversion of anomerle configuration.Key words: Chitinase; Lysozyme; Catalytic mechanism; Anomeri¢ configuration I, Introduction Chitinases (EC 3.2.1.14) are enzymes that catalyze the hydrolysis of 13-1,4 linkages in chitin, a homopolymer of N-acetyl-~glucosamine [1]. The substrates of chitinases and lysozymes are very similar. Indeed, some chitinases also hydrolyse bacterial cell walls [1,2].Plant chitinases are a structurally diverse group with respect to their physical properties, enzymatic activities and localization [3]. Some chitinases, especially in combination with [3-1,3 glucanases, inhibit fungal growth in vitro [4], substantiating the idea that these enzymes play an important role in plant defense against pathogens, Chitinases could also release elicitors from funlpd cell walls [5], Recently it was shown that root chitinases can differentially inactivate Nod-factors produced by certain P, ht=obtmn strains and therefore may determine the specificity of tl,e bacterium-host plant interaction as well as modulate the activity of Nod-factors [6]. Chitinase may also have an important function in development, as demonstrated by the ability of a chitinase to overcome a block in embryogenesis in a mutant cell line of carrot [7]. Plant chitinases have been divided into several classes based on amino acid sequence [8], In the classification system of glycosyl hydrolases, all chitinases are grouped into two families [9]. Class I, II, IV and V chitinases form the family 19, that is only known in plants. Family 18 includes all fungal, animal and bacterial chitinases as well as plant chitinases of classes III and VI.The hydrolytic action of glycosyl hydrolases can take place with either retention or inversion of the anomeric configuration. The hydrolytic mechanism of but a few chitinases has been analyzed. Two bacterial chitinases and hevamme, a plant chitinase/lysozyme displayed a retaining mechanism [10,11]. Hen egg white lysozyme (family 22) [12] is also a retaining enzyme, while T4 lysozyme (family 24) was recently found to invert the anomeric configuration [13]. Holm and Sander [14] recently proposed that a family 19 chitinase and several lysozymes descend from a common ancestor, based on a weak folding similarity and the similar function. Goose lysozyme (Ya...

Structure of Human Hyaluronidase-1, a Hyaluronan Hydrolyzing Enzyme Involved in Tumor Growth and Angiogenesis,

“…The double displacement results in retention of configuration at C1 (39,40). Tews et al proposed a substrate assisted mechanism for the chitin degrading enzymes in which the C2 N-acetyl's oxygen atom acts as a nucleophile (38).…”

A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037

“…This mechanism is supported by the fact that microbial family 18 chitinases are unable to cleave the glycosidic bonds of GlcN in partially acetylated chitosan. On the other hand, family 19 chitinases operate by an inverting mechanism (6,17) probably involving the direct attack of the sugar anomeric carbon by a nucleophilic water molecule (35). This mechanism, which has recently been shown to also prevail in a chitosanase (6), does not involve participation of the acetamido group at C-2 and, consequently, allows the hydrolysis of ␤-linked GlcNAc as well as GlcN.…”

Family 19 chitinases of Streptomyces species: characterization and distribution The GenBank accession numbers for the sequences determined in this work are AB031745–AB031757 inclusive.

“…In addition to the difference in 3D structure, chitinases of the two families show several important differences in their biochemical properties. For example, family 18 chitinases hydrolyse the glycosidic bond with retention of the anomeric configuration (Armand et al, 1994 ;Iseli et al, 1996), whereas family 19 chitinases hydrolyse with inversion (Ohno et al, 1996 ;Fukamizo et al, 1995). Family 18 chitinases are sensitive to allosamidin, but a family 19 chitinase from higher plants has been shown to be insensitive (Koga et al, 1987).…”