Difference between revisions of "Auxiliary Activity Family 10"

Revision as of 12:14, 15 January 2018

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Author: ^^^Vincent Eijsink^^^ and ^^^Gustav Vaaje-Kolstad^^^
Responsible Curator: ^^^Vincent Eijsink^^^

Auxiliary Activity Family 10
Clan	Structurally related to AA9 & AA11 & AA13
Mechanism	lytic oxidase
Active site residues	mononuclear copper ion
CAZy DB link
https://www.cazy.org/AA10.html

Substrate specificities

Figure 1. Hypothetical representation of the interaction between CBP21 and chitin (side view, left; top view, right) that highlights how the flat surface of CBP21 might fit the flat surface of a β-chitin crystal. Please note that this complex has not been determined by direct experimentation nor computational molecular modelling. The surfaces of residues known to interact with chitin [] are coloured magenta and the side chains of these residues are shown. In the side view some of the magenta surface is hidden by the white surface of other residues. This picture has been adapted from [].

Members of the AA10 family of lytic polysaccharide monooxygenases were previously classified as Carbohydrate Binding Module Family 33 members). They are known to cleave chitin [3, 4, 5] and cellulose [6, 7, 8]. Most characterized AA10 LPMO oxidize the C1 of the scissile glycosidic bond, but some also oxidize C4 [9, 10]. AA10 LPMOs are closely related to LPMOs in families AA11 and AA13, known to cleave chitin and starch, respectively. AA10 modules often occur in combination with additional modules, in particular carbohydrate-binding modules (CBMs), but also catalytic domains such as GH18 chitinases [2]. The CBMs contribute to substrate-binding and may also affect operational stability of the LPMO [10, 11].

Before proteins belonging to AA10 were identified as enzymes, they were generally known as chitin binding proteins (CBPs) and as carbohydrate-binding modules belonging to family CBM33. The reason for this was that most AA10s studied had been identified in chitinolytic systems such as that of Serratia marcescens [12, 13], several Streptomyces species [14, 15, 16], Bacillus amyloliquefaciens [17],Vibrio cholerae [18], Pseudomonas aeruginosa [19] and Lacotococcus lactis [20]. Upon their characterization no other function than substrate binding could be identified, thus the name "chitin binding protein" was coined. Substrates and potential substrates identified by binding studies include alpha-chitin [14, 15], beta-chitin [1, 13], both the alpha- and beta-chitin allomorphs [4, 17, 20] chitosan [16], cellulose [6, 21] and even bacterial and eptihelial cell surfaces where the binding interaction substrate has been suggested to be GlcNAc containing glycoproteins or proteoglycans [18, 22]. It should be noted that studies on AA10s prior to their identification as copper-dependent metalloenzymes were conducted in the absence of Cu(II), which may have had influence on the binding affinity and specificity of the enzyme.

As previously noted, AA10s exist both as single module entities and in multimodular forms. CBP21 from S. marcescens [3] and EfCBM33A from E. faecalis [4] are catalytically functional single-module AA10s that both bind well to chitin (CBP21 is specific for beta-chitin, whereas EfCBM33A binds to both alpha and beta-chitin). The cellulose targeting AA10 from S. coelicolor (CelS2) on the other hand, is bimodular and contains a cellulose binding CBM2 in addition to the catalytic AA10 module [6, 9, 11]. The V. cholerae AA10 is an elongated tetra-modular protein where the N-terminal catalytically actove AA10 module binds mucin, the two following modules bind bacterial cell walls and the C-terminal CBM5/12 bind chitin [18, 23].

The substrate binding surface of AA10s is flat and lacks the typical arrangement of aromatic amino acids that is common for carbohydrate binding proteins [1, 24]. It is therefore thought that substrate binding is predominantly mediated by hydrogen bonds, as is substantiated by a recent study of CelS2 [10]. The flat substrate binding surface seems optimal for binding the flat surface of crystalline carbohydrate structures like cellulose and chitin (Fig. 1).

Kinetics and Mechanism

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Catalytic Residues and copper coordination

Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace [25] (Fig. X) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion [3, 26], which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s [5, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36]. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial". One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water [37, 38]. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position [39, 40]. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. X) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s [39, 41] suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this position by a conserved alanine residue, the role of which has received considerable attention in the literature [9, 10, 37]. Current data do not point at a particular effect of the presence of this alanine nor of its exact position, which varies among AA10 LPMOs. More conserved feature; second shell, reson?

Three-dimensional structures

In 2005 the structure CBP21 from S. marcescens was solved and represents the first structure in the AA10 family 2BEM [1] and the first structure of an LPMO The CBP21 wild type structure has three molecules in the asymetric unit, of which only chain C shows electron density for a metal bound in the metal binding motif (modeled as a sodium ion, but, in retrospect this is probably a reduced copper ion with low occupancy). Later the same year the structure of the CBP21-Y54A mutant was solved (different crystal form and space group), showing two molecules in the asymetric unit with no trace of electron density for a metal ion bound in the active site 2BEN [42]. The second AA10 structure, one of two AA10 from Burkholderia pseudomallei 1710b (Uniprot ID: Q3JY22), was published in the PDB late in 2011 by Seattle Structural Genomics Center for Infectious Disease 3UAM. The structure contains five molecules in the asymetric unit that all have two amino acids from the signal peptide still attached to the N-terminus, most likely disrupting the active site. The third unique AA10 structure to be solved was GbpA from Vibrio cholerae O1 biovar El Tor str. N16961 2XWX [18]. GbpA is unique in the sense that it contains four discrete modules: a catalytically active N-terminal AA10 module, two modules likely invovled in binding to bacterial cell walls and a C-terminal CBM5/12. The published structure of GbpA only lacks the C-termainal CBM5/12 and is thus the first multimodular AA10 structure to be published. Shortly after the release of the GbpA, the structure of EfCBM33A from Enterococcus faecalis 4A02 [4] was published. The structure of EfCBM33A was solved at very high resolution (0.95Å), but similar to the other structures solved, no metal ion was observed bound in the active site. In 2012 the solution structure of CBP21 wild type (apo-form) was solved by NMR 2LHS [5], and this study also provided evidence for copper being the active site metal, as had been previously shown for AA11 LPMOs [25]. In spring 2013 the structure of the single AA10 harbored byBacillus amyloliquefaciens DSM7 (BaCBM33) was published [36]. The latter publication contained three structures: the apo-enzyme 2YOW and two structures containing a reduced copper ion bound to the active site 2YOY 2YOX, thus being the first AA10 structures with the copper ion (that is essential for activity) bound in the active site. The first structures of AA10 LPMOs known to be active on cellulose came in 2014 [9]. It is worth noting that the oxidation state of the copper ion is affected by the reducing power of the X-ray beam as dicussed by Hemsworth et al [37] and Gudmundsson et al [38].

Family Firsts

First AA10 protein identified: The first proteins studied from the AA10 family were all isolated and cloned from various Streptomyces strains, a major effort carried out by the Schrempf group of Osnabrück University. The first family AA10 protein to be isolated and characterized was CHB1 from Streptomyces olivaceoviridis a study published in 1994 [43]. CHB1 was shown to bind strongly to alpha-chitin and was also observed to bind to fungal hyphae. When these proteins originally were included into CAZy, they were classified as CBM33.
First demonstration of synergy between AA10 and canonical glycoside hydrolases: In 2005, Vaaje-Kolstad and co-workers showed that CBP21 from S. marcescens is able to increase the rate of chitin hydrolysis by a variety of chitinases, including all three S. marsescens GH18 chitinases and a GH19 chitinase from Streptomyces coelicolor [42]. It is worth noting that the title of the original publication erroneously qualified CBP21 as "non-catalytic"; this is due to how one was thinking about possible substrate-disrupting effects of CBMs at the time.
First demonstration of oxidative cleavage by an AA10 protein: Catalysis of lytic oxidation of a glycosidic bond by an AA10 enzyme was first shown for CBP21 in 2010, where oxidative cleavage of chitin was demonstrated [3]. This finding also represents the first demonstration of LPMO activity, regardless of AA family. Oxidative cleavage of cellulose by an AA10 was demonstrated by the same group in 2011 [6].
First 3-D structure: CBP21, the single AA10-type LPMO from the Gram negative bacterium Serratia marcescens, PDB ID 2BEM.

References

All Medline abstracts: PubMed

@@ Line 44: / Line 44: @@
 == Catalytic Residues and copper coordination ==
+Catalysis happens at the single catalytic copper ion that is coordinated by a so-called histidine brace <cite>Quinlan2011</cite> (Fig. X) that is strictly conserved in all LPMOs. Originally, there was some confusion about the nature of the metal ion <cite>Harris2010 Vaaje-Kolstad2010</cite>, which is due to the high copper affinity of LPMOs, which allows the enzymes to acquire copper even in the "absence" of copper. This issue was nicely settled in a seminal paper by Quinlan et al on AA9s <cite> and further elaborated for AA10s in subsequent publications <cite>Aachmann2012 Hemsworth2013</cite>. The two histidines making up the histidine brace coordinate the copper in three positions that, for practical reasons are often referred to as "equatorial". One of the histidines is the N-terminus of the mature protein and contributes with both its N-terminal amino group and a its imidazole side chain, whereas the other histidine contribute only with its side chain. In oxidized LPMOs, i.e. the Cu(II) form, the fourth equatorial position is occupied by a water <cite>Hemsworth2013-2 Gudmundsson2014</cite>. There are strong indications that the reactive oxygen species formed during catalysis emerges at this fourth equatorial position <cite>Frandsen2016 Bacik2017</cite>. The final two coordination positions, often referred to as axial, vary. The proximal axial coordination site (Fig. X) is occupied by a phenylalanine or a tyrosine. The functional implications, if any, of this difference remains unknown. It should be noted that, while the hydroxyl group of the tyrosine does point towards the copper ion, the distance to the copper is too long for a strong interaction. The distal, solvent-exposed axial position tends to be occupied by a water in oxidized LPMOs and recent studies on AA9s <cite>Courtade2016 Frandsen2016</cite> suggest that this water will be displaced by substrate binding. The copper ion is partly shielded from the solvent in this position by a conserved alanine residue, the role of which has received considerable attention in the literature <cite>Hemsworth2013-2 Forsberg2014 Forsberg2018</cite>. Current data do not point at a particular effect of the presence of this alanine nor of its exact position, which varies among AA10 LPMOs.
-Under construction. References <cite>Vaaje-Kolstad2010 Aachmann2012 Hemsworth2013 Hemsworth2013-2</cite>
+More conserved feature; second shell, reson?
 == Three-dimensional structures ==
@@ Line 86: / Line 86: @@
 #Gudmundsson2014 pmid=24828494
 #Quinlan2011 pmid=21876164
+#Harris2010 pmid=20230050
+#Frandsen2016 pmid=26928935
+#Bacik2017 pmid=28481095
+#Courtade2016 pmid=27152023
 </biblio>
 [[Category:Auxiliary Activity Families|AA010]]