Glycoside Hydrolase Family 19

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

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Glycoside Hydrolase Family GH19
Clan	none (lysozyme fold)
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 CBM [2, 3]. Detailed studies on GH19 chitinases from Streptomyces (class IV; [3]) and rice (Oryza sativa; Class I; [4]) have 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 [5] and HPLC [6]. Both structural characteristics (see below) and available biochemical data [3, 4] suggest that GH19 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. [7, 8]).

Catalytic Residues

Conserved residues surrounding the catalytic acid, Glu67, in the family 19 papaya chitinase (PDB ID 3cql). Amino acid side chains are shown in stick representation. Hydrogen bonds are shown as dashed lines with distances indicated in Å. A GlcNAc₂ unit binding in the -1 and +1 subsites is shown in narrow stick representation and an arrow indicates the position of the glycosidic oxygen. The sugar ring substituents are not shown in order to make the figure clearer. The structure of this complex comes from a model described in reference [9]. The picture is taken from reference [10].

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 [9, 11, 12, 13].

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 [14] (see Figure). Interestingly, a similarly complex electrostatic interaction network is present in family GH46 chitosanases [15, 16] 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 GH46 chitosanases and with lysozymes in families GH22, GH23 and GH24 of glycoside hydrolases [17]. For a long time, structural information for these chitinases was limited to the structures of two class II plant enzymes [11, 18]. Recently, the structures of bacterial family GH19 chitinases have become available ([13, 19]; class IV), as well as the structures of class I [20] and class IV [21] GH19 chitinases from plants.

<gallery> GH19both.PNG|thumb|left|300px|Structures of the barley GH19 chitinase (left; PDB ID 1cns)and ChiG from S. coelicolor (right; PDB ID 2cjl). The side chains of the catalytic acids are shown in green. Additionally, the side chains of several residues that are (putatively) involved in substrate binding and catalysis are shown in red and purple. The picture is taken from reference [13]. GH19suppos.PNG|thumb|right|300px|Structural superposition of barley GH19 chitinase (light blue ribbon) and ChiG from S. coelicolor (darker blue surface). The picture highlights structural differences between the enzymes.The side chains of the catalytic glutamates in the bacterial enzymes are shown and labelled. The picture is taken from reference [13]. </gallery The structures of bacterial GH19 chitinases revealed several differences from the previously reported plant structures ([13, 19]; 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 [20, 22].

There is no structural information for GH19 enzymes in complex with their substrate. In 2008, Huet et al published [9] 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 [23]

First stereochemistry determination

Yam chitinase, by NMR [5] and Bean chitinase, by HPLC [6].

First general base residue identification

Chitinase from barley; determination by site-directed mutagenesis [12], structural analysis [11] and modelling [24]. Additional support from structure determination and modelling of a papaya chitinase [9].

First general acid residue identification

Chitinase from barley; determination by site-directed mutagenesis [12], structural analysis [11] and modelling [24]. Additional support from structure determination and modelling of a papaya chitinase [9].

First 3-D structure

Barley chitinase [11].

References

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5. Fukamizo T, Koga D, Goto S Comparative biochemistry of chitinases - anomeric form of the reaction products Bioscience, Biotechnology & Biochemistry 1995, 59:311-313.

10. Hoell IA, Vaaje-Kolstad G, Eijsink VGH Structure and function of enzymes acting on chitin and chitosan Biotechnology and Genetic Engineering Reviews, Vol. 27, 2010, in press.