Difference between revisions of "Polysaccharide Lyase Family 1"

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Polysaccharide Lyase Family PL1
3-D Structure	β-helix
Mechanism	β-elimination
Charge neutraliser	calcium
Active site residues	known
CAZy DB link
https://www.cazy.org/PL1.html

Substrate specificities

Figure 1. Anti-β-elimination reaction catalysed by pectate lyase. R and R’ represent additional galacturonan residues

The PL1 family polysaccharide lyases (PL1) harness anti-β-elimination chemistry to cleave α-(1,4)-linked d-galacturonan to produce oligosaccharides with an unsaturated hexenuronic acid residue and a new reducing end (EC 4.2.2.2) [1, 2, 3]. Pectin lyases differ from pectate lyases as they are active against a substrate bearing methyl-ester groups at C6 (EC 4.2.2.10). The geometry of the α1,4-linkage facilitates anti-β-elimination about the C4-glycosidic oxygen bond.

Kinetics and Mechanism

The mechanism involves acidification of the C5 proton of the galacturonan residue binding to the +1 subsite of the enzyme, abstraction of this acidified proton, and subsequent leaving group elimination. The leaving group provides a new reducing end and the group from which it was eliminated has an unsaturated C4-C5 bond. The mechanism of proton abstraction from carbon acids is discussed by Gerlt and Gassman [4].

Catalytic Residues

Figure 2. Michaelis complex formed using Bacillus subtilis pectate lyase R279A mutant and trigalacturonic acid. The galacturonan residues are labelled according to the subsites they bind: -1, +1, +2. The catalytic arginine is modeled back in the position seen in the native structure and is close to the C5 proton abstracted. The two "catalytic" calcium-ions bound between enzyme and substrate carboxylates that acidify the C5 proton are Ca2 and Ca3.

Acidification of the C5 proton in the polysaccharide family 1 enzymes is by the binding of two “catalytic” calcium-ions which are bound only in the Michaelis complex and not to the free enzyme [5, 6, 7]. Abstraction of the C5 proton is by the catalytic arginine, the Brønstead base [5, 6, 7]. The Bacillus subtilis Michaelis complex is shown in Fig. 2. Protonation of the leaving group is probably by solvent water, though other possibilities have been discussed in the literature.

Three-dimensional structures

Pectate lyases PelC (endo-acting) [8] and PelE (exo-acting) [9] from Erwinia chrysanthemi, (2PEC) and endo-acting Bacillus subtilis pectate lyase [10] (1BN8). These PL1 structures are remarkable in showing the polypeptide chain folded into a right-handed superhelix comprising three β-strands per turn with turns stacking to form a domain of three parallel β-sheets. The structures showed that parallel β-sheets are stable in the absence of protecting α-helices and revealed remarkable side-chain stacks particularly in the hydrophobic interior. Structures of pectin lyase followed [11, 12], they are closely similar to pectate lyase have the arginine acting as Brønstead base but also have more hydrophobic active centres suitable for binding methylated pectin. The β-helix fold is conserved in other PL families including PL3 and PL9.

Family Firsts

First description of catalytic activity: Pectin trans-eliminase activity was first demonstrated in 1962 by Albersheim [1].
First catalytic base identification: Arginine was identified as the catalytic base from the complex of PelC with pentagalacturonate formed using the inactive mutant in which the arginine was substituted by lysine [5, 6]. The structure showed that the arginine would be in the correct position to abstract the C5 proton. Comparison of PL10 and PL1 Michaelis complexes cemented the role of the arginine as Brønstead base [13]. More detailed information on specificity emerged from a more recent study [7] which showed the central three subsites bound galacturonsyl-residues, but that the more remote subsites could tolerate methylated galacturonsyl-residues.
First catalytic divalent cation identification: The importance of acidifying the C5 proton by stabilizing the charge on the substrate carboxylate was acknowledged early though the conserved arginine was originally thought to fulfil this function [10]. It was later shown that two catalytic calcium-ions bound in the Michaelis complex acidify the C5 proton facilitating its abstraction [5, 6].
First 3-D structures: The first structures of PL1s reported were PelC [8] and PelE [9] from Erwinia chrysanthemi. The first PL1 structure from Bacillus subtilis was reported in 1994 [10].

References

All Medline abstracts: PubMed

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-* [[Author]]:
+* [[Author]]: [[User:Richard Pickersgill|Richard Pickersgill]]
-* [[Responsible Curator]]:  ^^^Richard Pickersgill^^^
+* [[Responsible Curator]]:  [[User:Richard Pickersgill|Richard Pickersgill]]
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 == Substrate specificities ==
-[[Image:Reaction.jpg|thumb|400px|'''Figure 1.''' Reaction catalysed by pectate lyase]]
+[[Image:Reaction.jpg|thumb|400px|'''Figure 1.''' ''Anti''-β-elimination reaction catalysed by pectate lyase. R and R’ represent additional galacturonan residues]]
-The PL1 family polysaccharide lyases harness ''anti''-β-elimination chemistry to cleave 1,4-linked α-D-galacturonan to produce oligosaccharides with an unsaturated hexenuronic acid residue and a new reducing end <cite>Albersheim1962, Edstrom1964a, Edstrom1964b</cite>.
+The PL1 family polysaccharide lyases ([http://www.cazy.org/PL1.html PL1]) harness ''anti''-β-elimination chemistry to cleave &alpha;-(1,4)-linked {{smallcaps|d}}-galacturonan to produce oligosaccharides with an unsaturated hexenuronic acid residue and a new reducing end ([http://www.enzyme-database.org/query.php?ec=4.2.2.2 EC 4.2.2.2]) <cite>Albersheim1962, Edstrom1964a, Edstrom1964b</cite>. Pectin lyases differ from pectate lyases as they are active against a substrate bearing methyl-ester groups at C6 ([http://www.enzyme-database.org/query.php?ec=4.2.2.10 EC 4.2.2.10]). The geometry of the α1,4-linkage facilitates ''anti''-β-elimination about the C4-glycosidic oxygen bond.
-In the figure (Fig. 1) showing pectate lyase activity R and R’ are additional galacturonan residues. Pectin lyases are active against a substrate bearing methyl-ester groups at C6.  The geometry of the α1,4-linkage facilitates ''anti''-β-elimination     about the C4-glycosidic oxygen bond.
 == Kinetics and Mechanism ==
@@ Line 40: / Line 39: @@
 == Catalytic Residues ==
-[[Image:PL1_active_site_crop.jpg|thumb|400px|'''Figure 2.''' Michaelis complex formed using B''acillus subtilis'' pectate lyase R279A mutant and trigalacturonic acid. The galacturonan residues are labelled according to the subsites they bind: -1, +1, +2.  The catalytic arginine is modelled back in the position seen in the native structure and is close to the C5 proton abstracted. The two "catalytic" calcium-ions bound between enzyme and substrate carboxylates that acidify the C5 proton are Ca2 and Ca3.]]
+[[Image:PL1_active_site_crop.jpg|thumb|400px|'''Figure 2.''' Michaelis complex formed using ''Bacillus subtilis'' pectate lyase R279A mutant and trigalacturonic acid. The galacturonan residues are labelled according to the subsites they bind: -1, +1, +2.  The catalytic arginine is modeled back in the position seen in the native structure and is close to the C5 proton abstracted. The two "catalytic" calcium-ions bound between enzyme and substrate carboxylates that acidify the C5 proton are Ca2 and Ca3.]]
-Acidification of the C5 proton in the polysaccharide family 1 enzymes is by the binding of two “catalytic” calcium-ions which are bound only in the Michaelis complex and not to the free enzyme [6-8]. Abstraction of the C5 proton is by the catalytic arginine, the Bronstead base [6-8].  The ''Bacillus subtilis'' Michaelis complex is shown in Fig. 2.  Protonation of the leaving group is probably by solvent water, though other possibilities have been discussed in the literature.
+Acidification of the C5 proton in the polysaccharide family 1 enzymes is by the binding of two “catalytic” calcium-ions which are bound only in the Michaelis complex and not to the free enzyme <cite>Scavetta1999, Herron2000, Seyedarabi2010</cite>. Abstraction of the C5 proton is by the catalytic arginine, the Brønstead base <cite>Scavetta1999, Herron2000, Seyedarabi2010</cite>.  The ''Bacillus subtilis'' Michaelis complex is shown in Fig. 2.  Protonation of the leaving group is probably by solvent water, though other possibilities have been discussed in the literature.
 == Three-dimensional structures ==
-Pectate lyases PelC (endo-acting) [9] and PelE (exo-acting)  [10] from ''Erwinia chrysanthemi'', and endo-acting ''Bacillus subtilis'' pectate lyase [11] (PDB codes: 2PEC, 1PCL, 1BN8). These structures were remarkable in showing the polypeptide chain folded into a right-handed superhelix comprising three β-strands per turn with turns stacking to form a domain of three parallel β-sheets.  The structures showed that parallel β-sheets are stable in the absence of protecting α-helices and revealed remarkable side-chain stacks particularly in the hydrophobic interior. Structures of pectin lyase followed [12, 13], they are closely similar to pectate lyase have the arginine acting as Bronstead base but also have more hydrophobic active centres suitable for binding methylated pectin.
+Pectate lyases PelC (endo-acting) <cite>Yoder1993</cite> and PelE (exo-acting)  <cite>Lietzke1994</cite> from ''Erwinia chrysanthemi'', ([{{PDBlink}}2pec 2PEC]) and endo-acting ''Bacillus subtilis'' pectate lyase <cite>Pickersgill1994</cite> ([{{PDBlink}}1bn8 1BN8]). These PL1 structures are remarkable in showing the polypeptide chain folded into a right-handed superhelix comprising three β-strands per turn with turns stacking to form a domain of three parallel β-sheets. The structures showed that parallel β-sheets are stable in the absence of protecting α-helices and revealed remarkable side-chain stacks particularly in the hydrophobic interior. Structures of pectin lyase followed <cite>Mayans1997, Vitali1998</cite>, they are closely similar to pectate lyase have the arginine acting as Brønstead base but also have more hydrophobic active centres suitable for binding methylated pectin. The β-helix fold is conserved in other PL families including [[PL3]] and [[PL9]].
 == Family Firsts ==
-;First description of catalytic activity: Pectin trans-eliminase activity was first demonstrated in 1962 by Albersheim [1].
+;First description of catalytic activity: Pectin trans-eliminase activity was first demonstrated in 1962 by Albersheim <cite>Albersheim1962</cite>.
-;First catalytic base identification: Arginine emerged as the catalytic base from the complex of PelC with pentagalacturonate formed using the inactive mutant in which the arginine was substituted by lysine [6, 7], the structure showed that the arginine would be in the correct position to abstract the C5 proton.  Comparison of PL10 and PL1 Michaelis complexes cemented the role of the arginine as Bronstead base [14].  More detailed information on specificity emerged from a more recent study [8] which showed the central three subsites bound galacturonsyl-residues, but that the more remote subsites could tolerate methylated galacturonsyl-residues.
+;First catalytic base identification: Arginine was identified as the catalytic base from the complex of PelC with pentagalacturonate formed using the inactive mutant in which the arginine was substituted by lysine <cite>Scavetta1999, Herron2000</cite>. The structure showed that the arginine would be in the correct position to abstract the C5 proton.  Comparison of PL10 and PL1 Michaelis complexes cemented the role of the arginine as Brønstead base <cite>Charnock2002</cite>.  More detailed information on specificity emerged from a more recent study <cite>Seyedarabi2010</cite> which showed the central three subsites bound galacturonsyl-residues, but that the more remote subsites could tolerate methylated galacturonsyl-residues.
-;First catalytic divalent cation identification: The importance of acidifying the C5 proton by stabilizing the charge on the substrate carboxylate was acknowledged early though the conserved arginine was originally thought to fulfil this function [11].  It was later shown that two catalytic calcium-ions bound in the Michaelis complex acidify the C5 proton facilitating its abstraction [6, 7].
+;First catalytic divalent cation identification: The importance of acidifying the C5 proton by stabilizing the charge on the substrate carboxylate was acknowledged early though the conserved arginine was originally thought to fulfil this function <cite>Pickersgill1994</cite>.  It was later shown that two catalytic calcium-ions bound in the Michaelis complex acidify the C5 proton facilitating its abstraction <cite>Scavetta1999, Herron2000</cite>.
-;First 3-D structures: ''Erwinia chrysanthemi'' pectate lyases: PelC [9]and PelE [10], and ''Bacillus subtilis'' pectate lyase [11].
+;First 3-D structures: The first structures of PL1s reported were PelC <cite>Yoder1993</cite> and PelE <cite>Lietzke1994</cite> from ''Erwinia chrysanthemi''. The first PL1 structure from ''Bacillus subtilis'' was reported in 1994 <cite>Pickersgill1994</cite>.
 == References ==
@@ Line 58: / Line 57: @@
 #Edstrom1964b pmid=14235514
 #Gerlt1993 pmid=8218268
+#Scavetta1999 pmid=10368179
+#Herron2000 pmid=10922032
+#Seyedarabi2010 pmid=20000851
+#Yoder1993 pmid=8502994
+#Lietzke1994 pmid=12232373
+#Pickersgill1994 pmid=7634076
+#Mayans1997 pmid=9195887
+#Vitali1998 pmid=9449837
+#Charnock2002 pmid=12221284
+</biblio>
-.            Gerlt, J.A. and P.G. Gassman, UNDERSTANDING ENZYME-CATALYZED PROTON ABSTRACTION FROM CARBON ACIDS - DETAILS OF STEPWISE MECHANISMS FOR BETA-ELIMINATION REACTIONS. Journal of the American Chemical Society, 1992. 114(15): p. 5928-5934.
-.            Gerlt, J.A. and P.G. Gassman, AN EXPLANATION FOR RAPID ENZYME-CATALYZED PROTON ABSTRACTION FROM CARBON ACIDS - IMPORTANCE OF LATE TRANSITION-STATES IN CONCERTED MECHANISMS. Journal of the American Chemical Society, 1993. 115(24): p. 11552-11568.
-.            Scavetta, R.D., et al., Structure of a plant cell wall fragment complexed to pectate lyase C. Plant Cell, 1999. 11(6): p. 1081-1092.
-.            Herron, S.R., et al., Structure and function of pectic enzymes: Virulence factors of plant pathogens. Proceedings of the National Academy of Sciences of the United States of America, 2000. 97(16): p. 8762-8769.
-.            Seyedarabi, A., et al., Structural Insights into Substrate Specificity and the anti beta-Elimination Mechanism of Pectate Lyase. Biochemistry, 2010. 49(3): p. 539-546.
-.            Yoder, M.D., N.T. Keen, and F. Jurnak, NEW DOMAIN MOTIF - THE STRUCTURE OF PECTATE LYASE-C, A SECRETED PLANT VIRULENCE FACTOR. Science, 1993. 260(5113): p. 1503-1507.
-.          Lietzke, S.E., et al., THE 3-DIMENSIONAL STRUCTURE OF PECTATE LYASE-E, A PLANT VIRULENCE FACTOR FROM ERWINIA-CHRYSANTHEMI. Plant Physiology, 1994. 106(3): p. 849-862.
-.          Pickersgill, R., et al., THE STRUCTURE OF BACILLUS-SUBTILIS PECTATE LYASE IN COMPLEX WITH CALCIUM. Nature Structural Biology, 1994. 1(10): p. 717-723.
-.          Mayans, O., et al., Two crystal structures of pectin lyase A from Aspergillus reveal a pH driven conformational change and striking divergence in the substrate-binding clefts of pectin and pectate lyases. Structure, 1997. 5(5): p. 677-689.
-.          Vitali, J., et al., The three-dimensional structure of Aspergillus niger pectin lyase B at 1.7-angstrom resolution. Plant Physiology, 1998. 116(1): p. 69-80.
-.          Charnock, S.J., et al., Convergent evolution sheds light on the anti-beta-elimination mechanism common to family 1 and 10 polysaccharide lyases. Proceedings of the National Academy of Sciences of the United States of America, 2002. 99(19): p. 12067-12072.
-</biblio>
 [[Category:Polysaccharide Lyase Families|PL001]]