High-resolution Crystal Structure of Arthrobacter aurescens Chondroitin AC Lyase: An Enzyme–Substrate Complex Defines the Catalytic Mechanism

Лунин, В.В.; Li, Yunge; Linhardt, Robert J.; Miyazono, H.; Kyogashima, Mamoru; Kaneko, T.; Bell, Alexander W.; Cygler, Mirosław

doi:10.1016/j.jmb.2003.12.071

Cited by 100 publications

(121 citation statements)

References 52 publications

(68 reference statements)

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“…Our study provides a representative structure of the PL12 family. Heparinase III shares a similar overall structure to heparinase II and Chondroitinase AC (ChonAC) which have been structurally well-characterized (Lunin et al, 2004;Shaya et al, 2006). These three proteins share a similar architecture composed of an (α/α) 5, 6 toroid and β-sandwich fold.…”

Section: Discussionmentioning

confidence: 98%

“…Two different approaches are employed to neutralize the negative charge on the carboxylic group. Pectin lyase utilizes a Ca 2+ ion to weaken the negative charge (Mayans et al, 1997) while others like HepII (Shaya et al, 2006) and ChonAC (Lunin et al, 2004) exert their asparagine residue to achieve the process. In our model, HepIII likely implements a similar strategy as ChonAC and HepII to accomplish the neutralization by employing its residue Asn260.…”

Section: Discussionmentioning

confidence: 99%

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Structural basis of heparan sulfate-specific degradation by heparinase III

Dong¹,

Lu²,

McKeehan³

et al. 2012

Protein Cell

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Heparinase III (HepIII) is a 73-kDa polysaccharide lyase (PL) that degrades the heparan sulfate (HS) polysaccharides at sulfate-rare regions, which are important co-factors for a vast array of functional distinct proteins including the well-characterized antithrombin and the FGF/FGFR signal transduction system. It functions in cleaving metazoan heparan sulfate (HS) and providing carbon, nitrogen and sulfate sources for host microorganisms. It has long been used to deduce the structure of HS and heparin motifs; however, the structure of its own is unknown. Here we report the crystal structure of the HepIII from Bacteroides thetaiotaomicron at a resolution of 1.6 Å. The overall architecture of HepIII belongs to the (α/α)5 toroid subclass with an N-terminal toroid-like domain and a C-terminal β-sandwich domain. Analysis of this high-resolution structure allows us to identify a potential HS substrate binding site in a tunnel between the two domains. A tetrasaccharide substrate bound model suggests an elimination mechanism in the HS degradation. Asn260 and His464 neutralize the carboxylic group, whereas Tyr314 serves both as a general base in C-5 proton abstraction, and a general acid in a proton donation to reconstitute the terminal hydroxyl group, respectively. The structure of HepIII and the proposed reaction model provide a molecular basis for its potential practical utilization and the mechanism of its eliminative degradation for HS polysaccarides.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Structural basis of heparan sulfate-specific degradation by heparinase III

Dong¹,

Lu²,

McKeehan³

et al. 2012

Protein Cell

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“…(ii) Aromatic residues such as tryptophan and phenylalanine undergo C-H/ interactions with the sugar rings of the substrate (32), thus optimizing its position with respect to the catalytically active residues to promote enzymatic activity (10). (iii) Polar residues such as tyrosine and asparagine form hydrogen bonds with various substituents on the substrate, further assisting in binding and positioning (4,12,14).…”

Section: Identification Of Putative Substrate-binding Residues In Smlmentioning

confidence: 99%

Insight into the Role of Substrate-binding Residues in Conferring Substrate Specificity for the Multifunctional Polysaccharide Lyase Smlt1473

MacDonald

Berger

2014

Journal of Biological Chemistry

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Background: A polysaccharide lyase (Smlt1473) from Stenotrophomonas maltophilia degrades multiple anionic polysaccharides in a pH-regulated manner. Results: Mutation of predicted substrate-binding residues significantly increased activity and specificity toward poly-␤-D-mannuronic acid or poly-␤-D-glucuronic acid. Conclusion: Substrate specificity is highly sensitive to conserved, charged, and aromatic residues flanking the active site. Significance: Smlt1473 may serve as a platform for the design of robust, highly specific polysaccharide lyases.

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“…Crystal structures of lyases belonging to families PL-1, -3, -4, -5, -6, -7, -8, -9, -10, -16, and -18 have been determined. These enzymes are grouped into six structural categories: (i) the parallel ␤-helix in PL-1 (13,14,16,17,55,56), -3 (57), -6 (58), and -9 (20); (ii) the ␣/␣-barrel in PL-5 (59) and -10 (60, 61); (iii) the ␣/␣-barrel plus anti-parallel ␤-sheet in PL-8 (29,(62)(63)(64)(65)(66); (iv) the ␤-sandwich plus ␤-sheet in PL-4 (33); (v) the ␤-jelly roll in PL-7 (27,67,68) and -18; and (vi) the triplestrand ␤-helix in PL-16 (69). The folds and substrates of polysaccharide lyases are summarized in Table 1.…”

Section: Resultsmentioning

confidence: 99%

“…These characteristics of lyases indicate that they share common structural features determining uronate recognition and reaction manner (␤-elimination). Crystal structures of polysaccharide lyases in 11 families have been determined thus far (Table 1), and structure and function relationships of enzymes such as lyases for pectate, pectin, alginate, chondroitin, hyaluronan, and xanthan are being analyzed (15,16,(27)(28)(29)(30)(31)(32). However, little knowledge has been accumulated on the mechanisms of substrate recognition and catalytic reaction of RG lyases.…”

mentioning

confidence: 99%

A Novel Structural Fold in Polysaccharide Lyases

Ochiai

Itoh

Maruyama

et al. 2007

Journal of Biological Chemistry

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Rhamnogalacturonan (RG) lyase produced by plant pathogenic and saprophytic microbes plays an important role in degrading plant cell walls. An extracellular RG lyase YesW from saprophytic Bacillus subtilis is a member of polysaccharide lyase family 11 and cleaves glycoside bonds in polygalacturonan as well as RG type-I through a ␤-elimination reaction. Crystal structures of YesW and its complex with galacturonan disaccharide, a reaction product analogue, were determined at 1.4 and 2.5 Å resolutions with final R-factors of 16.4% and 16.6%, respectively. The enzyme is composed of an eight-bladed ␤-propeller with a deep cleft in the center as a basic scaffold, and its structural fold has not been seen in polysaccharide lyases analyzed thus far. Structural analysis of the disaccharide-bound YesW and a site-directed mutagenesis study suggested that Arg-452 and Lys-535 stabilize the carboxyl group of the acidic polysaccharide molecule and Tyr-595 makes a stack interaction with the sugar pyranose ring. In addition to amino acid residues binding to the disaccharide, one calcium ion, which is coordinated by Asp-401, Glu-422, His-363, and His-399, may mediate the enzyme activity. This is, to our knowledge, the first report of a new structural category with a ␤-propeller fold in polysaccharide lyases and provides structural insights into substrate binding by RG lyase.Plant cell wall degradation is essential for plant pathogenic and saprophytic microbes to invade plants. The plant cell wall mainly consists of polysaccharides and proteins, with polysaccharides being more abundant. These polysaccharides are divided into three groups, cellulose, hemicellulose, and pectin (1). Pectin is more soluble in water than cellulose and hemicellulose, suggesting that this polysaccharide is the preferred target for plant-associated microbes. Pectin is further divided to three regions, i.e. polygalacturonan, rhamnogalacturonan type-I (RG-I), 2 and rhamnogalacturonan type-II (RG-II). In pectin molecules, polygalacturonan is present as a linear backbone, and RG-I and RG-II are attached to the backbone as branched chains (2-4). RG-I is a polymer with a disacchariderepeating unit consisting of L-rhamnopyranose and D-galactopyranouronic acid (GalA) as a main chain, and arabinans and galactans are attached to the main chain (5). RG-II has a backbone of polygalacturonan, and its side chains consist of a complex of ϳ30 monosaccharides, including rare sugars such as apiose and aceric acid (6). These plant cell wall polysaccharides become substrates to be degraded through reactions of polysaccharide hydrolases and/or lyases produced by plant pathogenic and saprophytic microbes. Hydrolases cleave glycoside bonds in polysaccharides via hydrolysis, and lyases do so by ␤-elimination reactions (7,8).The degradation pathway for the polygalacturonan region has been characterized extensively in microbes (9 -12). The structure and function relationships of polygalacturonan-degrading enzymes, including hydrolases and lyases have also been well documented (13)(...

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High-resolution Crystal Structure of Arthrobacter aurescens Chondroitin AC Lyase: An Enzyme–Substrate Complex Defines the Catalytic Mechanism

Cited by 100 publications

References 52 publications

Structural basis of heparan sulfate-specific degradation by heparinase III

Structural basis of heparan sulfate-specific degradation by heparinase III

Insight into the Role of Substrate-binding Residues in Conferring Substrate Specificity for the Multifunctional Polysaccharide Lyase Smlt1473

A Novel Structural Fold in Polysaccharide Lyases

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