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High-Resolution Crystal Structure of Arthrobacter Aurescens Chondroitin AC Lyase: an Enzyme–Substrate Complex Defines the Catalytic Mechanism

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High-Resolution Crystal Structure of Arthrobacter Aurescens Chondroitin AC Lyase: an Enzyme–Substrate Complex Defines the Catalytic Mechanism doi:10.1016/j.jmb.2003.12.071 J. Mol. Biol. (2004) 337, 367–386 High-resolution Crystal Structure of Arthrobacter aurescens Chondroitin AC Lyase: An Enzyme–Substrate Complex Defines the Catalytic Mechanism Vladimir V. Lunin1, Yunge Li1, Robert J. Linhardt2, Hirofumi Miyazono3 Mamoru Kyogashima3, Takuji Kaneko3, Alexander W. Bell4 and Miroslaw Cygler1* 1Biotechnology Research Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycan- Institute, National Research degrading enzymes that act as eliminases. Chondroitin lyase AC from Council of Canada, and Arthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfate Montre´al Joint Centre for and chondroitin 6-sulfate but not on dermatan sulfate. Like other chon- Structural Biology, Montre´al droitin AC lyases, it is capable of cleaving hyaluronan. Que´bec, 6100 Royalmount Ave. We have determined the three-dimensional crystal structure of Montre´al, Que´bec, Canada ArthroAC in its native form as well as in complex with its substrates H4P 2R2 (chondroitin 4-sulfate tetrasaccharide, CStetra and hyaluronan tetrasacchar- ide) at resolution varying from 1.25 A˚ to 1.9 A˚ . The primary sequence of 2Department of Chemistry ArthroAC has not been previously determined but it was possible to Division of Medicinal determine the amino acid sequence of this enzyme from the high- Chemistry and Department of resolution electron density maps and to confirm it by mass spectrometry. Chemical and Biochemical The enzyme–substrate complexes were obtained by soaking the substrate Engineering, The University of into the crystals for varying lengths of time (30 seconds to ten hours) and Iowa, 115 S. Grand Ave, PHAR flash-cooling the crystals. The electron density map for crystals soaked in S328, Iowa City, IA the substrate for as short as 30 seconds showed the substrate clearly and 52242-1112, USA indicated that the ring of central glucuronic acid assumes a distorted 3Central Research Laboratories boat conformation. This structure strongly supports the lytic mechanism Seikagaku Corporation, Tateno where Tyr242 acts as a general base that abstracts the proton from the C5 3-1253, Higashiyamato-shi position of glucuronic acid while Asn183 and His233 neutralize the charge Tokyo 207-0021, Japan on the glucuronate acidic group. Comparison of this structure with that of 4 chondroitinase AC from Flavobacterium heparinum (FlavoAC) provides an Montre´al Proteomics Network explanation for the exolytic and endolytic mode of action of ArthroAC 740 Dr Penfield Ave., Montre´al and FlavoAC, respectively. Que´bec, Canada H3A 1A4 Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved. Keywords: chondroitin lyase; chondroitinase AC; Arthrobacter aurescens; *Corresponding author substrate binding; catalytic mechanism Introduction ides, composed of disaccharide repeating units of a substituted glucosamine or galactosamine Glycosaminoglycans (GAGs) are the carbo- attached through (1,4) linkage to a uronic acid hydrate components of proteoglycans, which are molecule. These disaccharide units are linked (1,3) a major component of the extracellular matrix.1 or (1,4) into a polysaccharide chain.2 The glucosa- They are highly negatively charged polysacchar- mine/galactosamine units are sulfated extensively, and their synthesis requires the concerted action of a large number of enzymes.3,4 Supplementary data associated with this article can be Glycosaminoglycans are degraded enzymatically found at doi: 10.1016/j.jmb.2003.12.071 5 Abbreviations used: GAG, glycosaminoglycan; MS, by two types of enzymes, hydrolases and lyases. mass spectrometry. Hydrolases catalyze cleavage of the glycosyl- E-mail address of the corresponding author: oxygen bond by addition of water, producing [email protected] a saturated disaccharide. Lyases cleave the 0022-2836/$ - see front matter Crown Copyright q 2004 Published by Elsevier Ltd. All rights reserved. 368 Chondroitinase AC Crystal Structure and Mechanism oxygen–aglycone linkage through proton abstrac- as the complexes with chondroitin tetrasaccharide tion, producing an unsaturated disaccharide pro- (DUAp (1 ! 3)-b-D-GalpNAc4S (1 ! 4)-b-D-GlcAp duct with a double bond between C4 and C5. The (1 ! 3)-a,b-D-GalpNAc4S, where DUAp is the enzymatic mechanisms of hydrolases are well unsaturated sugar residue, 4-deoxy-a-L-threo-hex- understood and reactions proceed either according 4-enopyranosyluronic acid; GlcAp, glucopyrano- to the retaining or inverting mechanism.6 On the syluronic acid; GalpN, 2-deoxy-2-aminogalacto- other hand, the molecular details of the enzymatic pyranose; S, sulfate; and Ac, acetate) and mechanism of GAG lyases are still poorly under- hyaluronan tetrasaccharide substrates. stood. A chemically plausible mechanism for the b Despite the purification and characterization of elimination reaction has been proposed;7 however, ArthroAC many years ago13,17,18 and its extensive the constitution of the active site and the roles of use as an analytical tool in glycosaminoglycan individual amino acids are not clear. A number of analysis, this enzyme has not been cloned and its bacterial species synthesize GAG lyases, enzymes amino acid sequence has not been determined, used to degrade and utilize glycosaminoglycans although its amino acid composition and carbo- as a source of carbon in the bacterium’s natural hydrate content were reported.18 We obtained environment.5,8 Polysaccharide lyases with known crystals that diffract up to 1.25 A˚ resolution. The three-dimensional structures fall into two architec- high-resolution data led to high-quality electron tures: the right-handed parallel b-helix (pectate/ density maps of native enzyme and several com- pectin lyases, chondroitinase B, rhamnoglucuronan plexes, and allowed us to deduce confidently the lyase) and (a/a)n toroid (n ¼ 5 for Flavobacterium amino acid sequence for 99% of the amino acid heparinum chondroitin AC and chondroitin ABC residues and to propose the molecular details of lyases, bacterial hyaluronate lyases, xanthan lyase, the catalytic mechanism, which is common to and n ¼ 6 for alginate lyases). A catalytic mechan- FlavoAC and hyaluronate lyases. Here, we follow ism has been proposed for pectate lyases, Ca2þ- the nomenclature introduced by Davies et al.19 and dependent enzymes,9 but it remains to be seen if it designate the sugars on the reducing end of the applies to other lyases having the b-helix topology. break with a þ sign and the sugars on the non- Several plausible mechanisms have been proposed reducing end with a 2 sign. In this nomenclature, for the lyases with the (a/a)5 toroidal fold with the enzymes break the bond between sugars 21 a histidine or a tyrosine residue in the role of a and þ1, the latter being an uronic acid. general base abstracting the proton from the C5 atom of glucuronic acid, and a tyrosine or an argin- ine residue acting as a general acid donating a pro- Results and Discussion ton to the bridging O4 atom.10,11 However, there is insufficient evidence to indicate which of these Amino acid sequence and its conservation proposed mechanisms is utilized by the enzymes. The nature of the group presumed to be necessary The molecular mass of the entire molecule was to neutralize the charge of the glucuronic acid measured by ion spray mass spectrometry. Two carboxylic group is not clear, since these enzymes species were present with molecular masses of do not require Ca2þ and there is no positively 79,502 Da and 79,840 Da. The greater mass corre- charged group in the vicinity of the uronic acid, as sponds well to the molecular mass of 79,785 Da expected from the accepted chemical mechanism.7 (average mass) calculated from the amino acid Glycosaminoglycan-degrading enzymes with sequence. Based on the analysis of MS/MS spectra, defined specificity have found widespread appli- we believe that the smaller mass corresponds to cations as analytical tools for the analysis of the the fragment missing three N-terminal amino acid structure of glycosaminoglycans and other poly- residues. Such a good agreement between the pre- saccharides.12 Chondroitin AC lyases are used fre- dicted and measured molecular mass indicated quently for this purpose. These enzymes cleave that (1) no major assignment errors were made the glycosidic bond on the non-reducing end of an and (2) no glycosylation or other modification uronic acid and use as a substrate either chondroi- were present (FlavoAC is glycosylated).16 tin 4-sulfate or chondroitin 6-sulfate but not Peptides extracted from an in-gel trypsin digest dermatan sulfate. They display a varied degree of purified A. aurescens chondroitin AC lyase were of activity toward hyaluronan.13 Enzymes from analyzed by LC-QToF mass spectrometry as two sources, chondroitin AC lyase from Arthrobacter described in Materials and Methods. The resulting aurescens (ArthroAC) and from F. heparinum peaklist of fragmentation spectra was matched in- (FlavoAC), are commercially available (Seikagaku house against the sequence deduced from the crys- Corporation) and used frequently. The latter tal structure employing Mascot (MatrixScience) enzyme has been cloned and overexpressed in software.20 Matched tandem MS spectra were con- F. heparinum14 and in Escherichia coli.15 We pre- firmed manually and the remaining spectra were viously determined the three-dimensional struc- interpreted manually. This process was repeated ture of this enzyme on its own16 and in complex several times, using the two sets of experimental with several dermatan sulfate oligosaccharides.10 data iteratively to determine the optimal sequence Here, we present the three-dimensional structure of the lyase (Supplementary Material). of chondroitin AC lyase from A. aurescens as well The results of MS/MS analysis of 202 tandem Chondroitinase AC Crystal Structure and Mechanism 369 mass spectra covering 88.5% of the entire lyase 1F1S22 and Bacillus sp. xanthan lyase 1J0M.23 ArthroAC sequence (see Materials and Methods) Structure-based alignment of their sequences is confirmed the amino acid sequence identified shown in Figure 1. The residues important for the from electron density maps.
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