Glycoside Hydrolase Family 19

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• Author: ^^^Vincent Eijsink^^^
• Responsible Curator: ^^^Vincent Eijsink^^^

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Glycoside Hydrolase Family GHnn
Clan	GH-x
Mechanism	inverting
Active site residues	known
CAZy DB link
http://www.cazy.org/fam/GH19.html

Substrate specificities

Glycoside hydrolases of family 19 hydrolyze glycoside bonds in chitin, an insoluble polymer of beta-1,4-linked N-acetyl-D-glucosamine (GlcNAc) and are thus referred to as chitinases (EC 3.2.1.14). These enzymes were originally identified in plants. In an older classification system for plant chitinases, comprising both GH18 and GH19 chitinases, family 19 enzymes comprise classes I, II and IV. In 1996, the first bacterial family 19 chitinase was described [1]. In addition to cleaving chitin, GH19 chitinases cleave soluble oligomers of beta-1,4-linked N-acetyl-D-glucosamine. For some plant enzymes lysozyme activity has been demonstrated. Currently available data suggest that GH19 enzymes are not particularly effective in degrading crystalline chitin (compared to certain members of the GH18 chitinase family), especially enzymes that lack CBMs. On the other hand GH19 enzymes are highly active on chitosans (= partially deacetylated chitin) with high degrees of acetylation, even if they lack a ChBD [2, 3]. Detailed studies on GH19 chitinases from Streptomyces (class IV; [3]) and rice (Oryza sativa; Class I; [4]) have both revealed that productive binding requires a GlcNAc to be bound in subsites -2 and +1, whereas deacetylated GlcNAc (GlcN) is tolerated in subsites -1 and +2.

Kinetics and Mechanism

Family 19 enzymes employ an inverting mechanism, as determined by NMR (Fukamizo etal.,1995) and HPLC (Iseli et al., 1996). Both structural characteristics (see below) and available biochemical data (Sasaki et al., 2006; Heggset et al., 2009) suggest that GH 19 chitinases are non-processive endo-acting enzymes. Kinetic data for the conversion of polymeric and oligomeric substrates have been described in several studies. In some studies, kinetic data have been used to derive subsite binding affinties (e.g. Honda & Fukamizo, 1998; Sasaki et al., 2003).

Catalytic Residues

The catalytic residues are two glutamates. Although there still is limited structural information underpinning details of the inverting catalytic mechanism, there is considerable support for the notion that a glutamate located at the end of the third alpha helix (Glu 67 in the barley enzyme) acts as the catalytic acid, whereas another glutamate located in a more variable loop-like structure (Glu89 in the barley enzyme) acts as the catalytic base (Hart et al, 1993; Andersen et al., 1997; Hoell et al., 2006; Huet et al., 2008).

It has been shown that at least two more conserved charged residues are crucial for catalysis. These residues, Glu203 and Arg215 in barley chitinase, form a triad together with the catalytic acid Glu67 (Ohnishi et al., 2005) (see Figure). Interestingly, a similarly complex electrostatic interaction network is present in family 46 chitosanases (Fukamizo et al., 2000; Lacombe-Harvey et al., 2009) with whom the family 19 enzymes share some overall structural similarity (see below).

Three-dimensional structures

The catalytic domains of family 19 chitinases have a lysozyme-like fold with rather shallow substrate-binding grooves that are not particularly rich in aromatic residues (see Figure). The catalytic domains of family 19 chitinases share a common fold with family 46 chitosanases and with lysozymes in families 22, 23 and 24 of glycoside hydrolases (Holm and Sander, 1994; Hart et al., 1995; Monzingo et al., 1996). For a long time, structural information for these chitinases was limited to the structures of two class II plant enzymes (Hart et al., 1993; Hahn et al., 2000). Recently, the structures of bacterial family 19 chitinases have become available (Hoell et al., 2006; Kezuka et al., 2006, class IV), as well as the structures of class I (Ubhayasekera et al. 2007) and class IV (Ubhayasekera et al. 2009) GH19 chitinases from plants.

The structures of bacterial GH19 chitinases revealed several differences from the previously reported plant structures (Hoell et al., 2006; Kezuka et al., 2006; see Figure). Compared to plant enzymes, the bacterial enzymes lack a C-terminal extension and three loops, some of which are thought to be flexible (Ubhayasekera et al., 2007; Fukamizo et al., 2009).

There is no structural information for GH19 enzymes in complex with their substrate. In 2008, Huet et al published the structure of a complex of papaya family 19 chitinase with GlcNAc units bound in the -2 and +1 subsites. This structure has been used to build a plausible model of a complex with (GlcNAc)4. This is the first structure (half experimental, half modeled) of an enzyme-substrate complex.

Family Firsts

First primary sequence determination: Bean leaf chitinase (Broglie et al., 1986)

First stereochemistry determination: Yam chitinase, by NMR (Fukamizo et al., 1995) and Bean chitinase, by HPLC (Iseli et al., 1996)

First general base residue identification: Chitinase from barley; determination by site-directed mutagenesis (Andersen et al., 1997), structural analysis (Hart et al., 1993) and modelling (Brameld and Goddard, 1998). Additional support from structure determination and modelling of a papaya chitinase (Huet et al., 2008).

First general acid residue identification: Chitinase from barley; determination by site-directed mutagenesis (Andersen et al., 1997), structural analysis (Hart et al., 1993) and modelling (Brameld and Goddard, 1998). Additional support from structure determination and modelling of a papaya chitinase (Huet et al., 2008).

First 3-D structure: Barley chitinase (Hart et al., 1993).

References

1. Ohno T, Armand S, Hata T, Nikaidou N, Henrissat B, Mitsutomi M, and Watanabe T. (1996). A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037. J Bacteriol. 1996;178(17):5065-70. DOI:10.1128/jb.178.17.5065-5070.1996 |
2. Kawase T, Yokokawa S, Saito A, Fujii T, Nikaidou N, Miyashita K, and Watanabe T. (2006). Comparison of enzymatic and antifungal properties between family 18 and 19 chitinases from S. coelicolor A3(2). Biosci Biotechnol Biochem. 2006;70(4):988-98. DOI:10.1271/bbb.70.988 |
3. Heggset EB, Hoell IA, Kristoffersen M, Eijsink VG, and Vårum KM. (2009). Degradation of chitosans with chitinase G from Streptomyces coelicolor A3(2): production of chito-oligosaccharides and insight into subsite specificities. Biomacromolecules. 2009;10(4):892-9. DOI:10.1021/bm801418p |
4. Sasaki C, Vårum KM, Itoh Y, Tamoi M, and Fukamizo T. (2006). Rice chitinases: sugar recognition specificities of the individual subsites. Glycobiology. 2006;16(12):1242-50. DOI:10.1093/glycob/cwl043 |