Difference between revisions of "Glycoside Hydrolase Family 19"

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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/GH19.html

Substrate specificities

Glycoside hydrolases of family 19 are endo-acting enzymes that hydrolyze glycosidic bonds within chitin, an insoluble polymer of β-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 β-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 analysis[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 []. The picture is taken from reference [].

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 general acid, whereas another glutamate located in a more variable loop-like structure (Glu89 in the barley enzyme) acts as the catalytic general 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.

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.

Structure images

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 [].

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 [].

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

All Medline abstracts: PubMed

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 {| {{Prettytable}}
 |-
-|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GHnn'''
+|{{Hl2}} colspan="2" align="center" |'''Glycoside Hydrolase Family GH19'''
 |-
 |'''Clan'''
-|GH-x
+|none<br>(lysozyme fold)
 |-
 |'''Mechanism'''
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 |{{Hl2}} colspan="2" align="center" |'''CAZy DB link'''
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-| colspan="2" |http://www.cazy.org/fam/19nn.html
+| colspan="2" |{{CAZyDBlink}}GH19.html
 |}
 </div>
 <!-- This is the end of the table -->
 == Substrate specificities ==
+[[Glycoside hydrolases]] of family 19 are [[endo]]-acting enzymes that hydrolyze glycosidic bonds within chitin, an insoluble polymer of &beta;-1,4-linked ''N''-acetyl-D-glucosamine (GlcNAc) and are thus referred to as chitinases (EC [{{EClink}}3.2.1.14 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 <cite>Ohno1996</cite>. 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 <cite>Kawase2006 Heggset2009</cite>. Detailed studies on GH19 chitinases from ''Streptomyces'' (class IV; <cite>Heggset2009</cite>) and rice (''Oryza sativa''; Class I; <cite>Sasaki2006</cite>) 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.
-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 [Ohno et al., 1996]. 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 (Kawase et al., 2006; Heggset et al., 2009). Detailed studies on GH19 chitinases from ''Streptomyces'' (class IV; Heggset et al., 2009) and rice (''Oryza sativa''; Class I; Sasaki et al., 2006) 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 <cite>Fukamizo1995</cite> and HPLC analysis<cite>Iseli1996</cite>. Both structural characteristics (see below) and available biochemical data <cite>Sasaki2006 Heggset2009</cite> 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. <cite>Honda1998 Sasaki2003</cite>).
-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 ==
+[[Image:GH19_detail.PNG|thumb|right|300px|'''Conserved residues surrounding the catalytic acid, Glu67, in the family 19 papaya chitinase (PDB ID [{{PDBlink}}3cql 3cql]).''' Amino acid side chains are shown in stick representation. Hydrogen bonds are shown as dashed lines with distances indicated in Å. A GlcNAc<sub>2</sub> 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 <cite>Huet2008</cite>. The picture is taken from reference <cite>Hoell2010</cite>.]]
+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 [[general acid]], whereas another glutamate located in a more variable loop-like structure (Glu89 in the barley enzyme) acts as the catalytic [[general base]]<cite>Hart1993 Andersen1997 Hoell2006 Huet2008</cite>.
-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 <cite>Ohnishi2005</cite> (see Figure). Interestingly, a similarly complex electrostatic interaction network is present in family [[GH46]] chitosanases <cite>Fukamizo2000 Lacombe2009</cite> with whom the family 19 enzymes share some overall structural similarity (see below).
-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 [[GH46]] chitosanases and with lysozymes in families [[GH22]], [[GH23]] and [[GH24]] of glycoside hydrolases <cite>Monzingo1996</cite>. For a long time, structural information for these chitinases was limited to the structures of two class II plant enzymes <cite>Hart1993 Hahn2000</cite>. Recently, the structures of bacterial family [[GH19]] chitinases have become available (<cite>Hoell2006 Kezuka2006</cite>; class IV), as well as the structures of class I <cite>Ubhayasekera2007</cite> and class IV <cite>Ubhayasekera2009</cite> GH19 chitinases from plants.
+The structures of bacterial GH19 chitinases revealed several differences from the previously reported plant structures (<cite>Hoell2006 Kezuka2006</cite>; 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 <cite>Ubhayasekera2007 Fukamizo2009</cite>.
-== Three-dimensional structures ==
+There is no structural information for GH19 enzymes in complex with their substrate. In 2008, Huet et al published <cite>Huet2008</cite> 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)<sub>4</sub>. This is the first structure (half experimental, half modeled) of an enzyme-substrate complex.
-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.
+=== Structure images ===
+[[Image:GH19both.PNG|thumb|left|560px|'''Structures of the barley GH19 chitinase (left; PDB ID [{{PDBlink}}1cns 1cns])and ChiG from ''S. coelicolor'' (right; PDB ID [{{PDBlink}}2cjl 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 <cite>Hoell2006</cite>.]]
-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).
+[[Image:GH19suppos.PNG|thumb|left|330px|'''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 <cite>Hoell2006</cite>.]]
-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.
+<br style="clear: both" />
 == Family Firsts ==
-First primary sequence determination:
+;First primary sequence determination: Bean leaf chitinase <cite>Broglie1986</cite>
-Bean leaf chitinase (Broglie et al., 1986)
-First stereochemistry determination:
+;First stereochemistry determination: Yam chitinase, by NMR <cite>Fukamizo1995</cite> and Bean chitinase, by HPLC <cite>Iseli1996</cite>.
-Yam chitinase, by NMR (Fukamizo et al., 1995) and Bean chitinase, by HPLC (Iseli et al., 1996)
-First general base residue identification:
+;First [[general base]] residue identification: Chitinase from barley; determination by site-directed mutagenesis <cite>Andersen1997</cite>, structural analysis <cite>Hart1993</cite> and modelling <cite>Brameld1998</cite>. Additional support from structure determination and modelling of a papaya chitinase <cite>Huet2008</cite>.
-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:
+;First [[general acid]] residue identification: Chitinase from barley; determination by site-directed mutagenesis <cite>Andersen1997</cite>, structural analysis <cite>Hart1993</cite> and modelling <cite>Brameld1998</cite>. Additional support from structure determination and modelling of a papaya chitinase <cite>Huet2008</cite>.
-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:
+;First 3-D structure: Barley chitinase <cite>Hart1993</cite>.
-Barley chitinase (Hart et al., 1993).
 == References ==
 <biblio>
+#Ohno1996 pmid=8752320
+#Kawase2006 pmid=16636468
+#Heggset2009 pmid=19222164
+#Sasaki2006 pmid=16957091
+#Fukamizo1995 Fukamizo T, Koga D, Goto S ''Comparative biochemistry of chitinases - anomeric form of the reaction products'' Bioscience, Biotechnology & Biochemistry 1995, 59:311-313.
+#Hoell2010 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.
+#Iseli1996 pmid=8612749
+#Honda1998 pmid=9774706
+#Sasaki2003 pmid=12825688
+#Hart1993 pmid=8421299
+#Andersen1997 pmid=9148754
+#Hoell2006 pmid=17010167
+#Huet2008 pmid=18636748
+#Ohnishi2005 pmid=16272567
+#Fukamizo2000 pmid=10829022
+#Lacombe2009 pmid=19143844
+#Monzingo1996 pmid=8564539
+#Hahn2000 pmid=10957628
+#Kezuka2006 pmid=16516924
+#Ubhayasekera2007 pmid=17608716
+#Ubhayasekera2009 pmid=19629717
+#Fukamizo2009 pmid=19332152
+#Broglie1986 pmid=2428042
+#Brameld1998 pmid=9539727
 </biblio>
 [[Category:Glycoside Hydrolase Families|GH019]]