Structural Determinants Responsible for Substrate Recognition and Mode of Action in Family 11 Polysaccharide Lyases
2009; Elsevier BV; Volume: 284; Issue: 15 Linguagem: Inglês
10.1074/jbc.m807799200
ISSN1083-351X
AutoresAkihito Ochiai, Takafumi Itoh, Bunzo Mikami, Wataru Hashimoto, Kousaku Murata,
Tópico(s)Polysaccharides Composition and Applications
ResumoA saprophytic Bacillus subtilis secretes two types of rhamnogalacturonan (RG) lyases, endotype YesW and exotype YesX, which are responsible for an initial cleavage of the RG type I (RG-I) region of plant cell wall pectin. Polysaccharide lyase family 11 YesW and YesX with a significant sequence identity (67.8%) cleave glycoside bonds between rhamnose and galacturonic acid residues in RG-I through a β-elimination reaction. Here we show the structural determinants for substrate recognition and the mode of action in polysaccharide lyase family 11 lyases. The crystal structures of YesW in complex with rhamnose and ligand-free YesX were determined at 1.32 and 1.65 Å resolution, respectively. The YesW amino acid residues such as Asn152, Asp172, Asn532, Gly533, Thr534, and Tyr595 in the active cleft bind to rhamnose molecules through hydrogen bonds and van der Waals contacts. Other rhamnose molecules are accommodated at the noncatalytic domain far from the active cleft, revealing that the domain possibly functions as a novel carbohydrate-binding module. A structural comparison between YesW and YesX indicates that a specific loop in YesX for recognizing the terminal saccharide molecule sterically inhibits penetration of the polymer over the active cleft. The loop-deficient YesX mutant exhibits YesW-like endotype activity, demonstrating that molecular conversion regarding the mode of action is achieved by the addition/removal of the loop for recognizing the terminal saccharide. This is the first report on a structural insight into RG-I recognition and molecular conversion of exotype to endotype in polysaccharide lyases. A saprophytic Bacillus subtilis secretes two types of rhamnogalacturonan (RG) lyases, endotype YesW and exotype YesX, which are responsible for an initial cleavage of the RG type I (RG-I) region of plant cell wall pectin. Polysaccharide lyase family 11 YesW and YesX with a significant sequence identity (67.8%) cleave glycoside bonds between rhamnose and galacturonic acid residues in RG-I through a β-elimination reaction. Here we show the structural determinants for substrate recognition and the mode of action in polysaccharide lyase family 11 lyases. The crystal structures of YesW in complex with rhamnose and ligand-free YesX were determined at 1.32 and 1.65 Å resolution, respectively. The YesW amino acid residues such as Asn152, Asp172, Asn532, Gly533, Thr534, and Tyr595 in the active cleft bind to rhamnose molecules through hydrogen bonds and van der Waals contacts. Other rhamnose molecules are accommodated at the noncatalytic domain far from the active cleft, revealing that the domain possibly functions as a novel carbohydrate-binding module. A structural comparison between YesW and YesX indicates that a specific loop in YesX for recognizing the terminal saccharide molecule sterically inhibits penetration of the polymer over the active cleft. The loop-deficient YesX mutant exhibits YesW-like endotype activity, demonstrating that molecular conversion regarding the mode of action is achieved by the addition/removal of the loop for recognizing the terminal saccharide. This is the first report on a structural insight into RG-I recognition and molecular conversion of exotype to endotype in polysaccharide lyases. Carbohydrate-active enzymes such as glycoside hydrolases (GHs) 2The abbreviations used are: GH, glycoside hydrolase; PL, polysaccharide lyase; RG, rhamnogalacturonan; Rha, rhamnose; GalA, galacturonic acid; RG chain, RG-I main chain; ΔGalA-Rha, unsaturated galacturonyl rhamnose; CBM, carbohydrate-binding module. (1Coutinho P.M. Henrissat B. Gilbert H.J. Davies G. Henrissat B. Svensson B. Carbohydrate-active Enzymes: An Integrated Database Approach. The Royal Society of Chemistry, Cambridge1999Google Scholar), polysaccharide lyases (PLs), glycosyl transferases, and carbohydrate esterases are categorized into over 200 families based on their amino acid sequences in the Carbohydrate-Active enZymes (CAZy) data base (1Coutinho P.M. Henrissat B. Gilbert H.J. Davies G. Henrissat B. Svensson B. Carbohydrate-active Enzymes: An Integrated Database Approach. The Royal Society of Chemistry, Cambridge1999Google Scholar). Lyases are classified into 18 PL families. PLs commonly recognize uronic acid residues in polysaccharides, catalyze a β-elimination reaction, and produce unsaturated saccharides with C=C double bonds in uronic acid residues at the newly formed nonreducing terminus. The crystal structures of PLs in 12 families have been determined thus far, and the structure and functional relationships of enzymes such as lyases for polygalacturonan, alginate, chondroitin, hyaluronan, and xanthan have been demonstrated (2Yoder M.D. Jurnak F. Plant Physiol. 1995; 107: 349-364Crossref PubMed Scopus (61) Google Scholar, 3Mayans O. Scott M. Connerton I. Gravesen T. Benen J. Visser J. Pickersgill R. Jenkins J. Structure. 1997; 5: 677-689Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 4Yamasaki M. Ogura K. Hashimoto W. Mikami B. Murata K. J. Mol. Biol. 2005; 352: 11-21Crossref PubMed Scopus (91) Google Scholar, 5Yoon H.-J. Hashimoto W. Miyake O. Murata K. Mikami B. J. Mol. Biol. 2001; 307: 9-16Crossref PubMed Scopus (78) Google Scholar, 6Lunin V.V. Li Y. Linhardt R.J. Miyazono H. Kyogashima M. Kaneko T. Bell A.W. Cygler M. J. Mol. Biol. 2004; 337: 367-386Crossref PubMed Scopus (93) Google Scholar, 7Shaya D. Tocilj A. Li Y. Myette J. Venkataraman G. Sasisekharan R. Cygler M. J. Biol. Chem. 2006; 281: 15525-15535Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 8Maruyama Y. Hashimoto W. Mikami B. Murata K. J. Mol. Biol. 2005; 350: 974-986Crossref PubMed Scopus (28) Google Scholar, 9Maruyama Y. Mikami B. Hashimoto W. Murata K. Biochemistry. 2007; 46: 781-791Crossref PubMed Scopus (18) Google Scholar, 10Ogura K. Yamasaki M. Mikami B. Hashimoto W. Murata K. J. Mol. Biol. 2008; 380: 373-385Crossref PubMed Scopus (60) Google Scholar). On the other hand, little knowledge has been accumulated on the mechanisms of substrate recognition and catalytic reaction in lyases acting on the rhamnogalacturonan (RG) region of pectin. Pectin, the major component of the plant cell wall, is divided into three regions, i.e. polygalacturonan, RG type I (RG-I), and RG 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 (11Darvill A.G. McNeil M. Albersheim P. Plant Physiol. 1978; 62: 418-422Crossref PubMed Google Scholar, 12Thakur B.R. Singh R.K. Handa A.K. Crit. Rev. Food Sci. Nutr. 1997; 37: 47-73Crossref PubMed Scopus (1154) Google Scholar, 13McNeil M. Darvill A.G. Fry S.C. Albersheim P. Annu. Rev. Biochem. 1984; 53: 625-663Crossref PubMed Scopus (800) Google Scholar). RG-I is a polymer with a disaccharide-repeating unit consisting of l-rhamnopyranose (Rha) and d-galactopyranouronic acid (GalA) as the main chain, and arabinans and galactans are attached to the main chain (14McNeil M. Darvill A.G. Albersheim P. Plant Physiol. 1980; 66: 1128-1134Crossref PubMed Google Scholar). RG-II has a backbone of polygalacturonan, and its side chains consist of a complex of about 30 monosaccharides, including rare sugars such as apiose and aceric acid (15O'Neill M.A. Warrenfeltz D. Kates K. Pellerin P. Doco T. Darvill A.G. Albersheim P. J. Biol. Chem. 1996; 271: 22923-22930Abstract Full Text Full Text PDF PubMed Scopus (428) Google Scholar). RG lyases are responsible for cleaving the α-1,4 bonds of the RG-I main chain (RG chain) through the β-elimination reaction (Fig. 1A) and belong to PL families 4 and 11, which mainly contain fungal and bacterial enzymes, respectively. Recently, we have reported the enzymatic route for degradation of the RG chain in a saprophytic Bacillus subtilis strain 168 (16Ochiai A. Itoh T. Kawamata A. Hashimoto W. Murata K. Appl. Environ. Microbiol. 2007; 73: 3803-3813Crossref PubMed Scopus (61) Google Scholar). This bacterium secretes two types of PL family 11 RG lyases, YesW and YesX, extracellularly. YesW cleaves the glycoside bond of the RG chain endolytically, and the resultant oligosaccharides are subsequently converted to disaccharides, unsaturated galacturonyl rhamnose (ΔGalA-Rha), through the exotype YesX reaction (16Ochiai A. Itoh T. Kawamata A. Hashimoto W. Murata K. Appl. Environ. Microbiol. 2007; 73: 3803-3813Crossref PubMed Scopus (61) Google Scholar) (Fig. 1). The crystal structures of YesW and its complex with GalA disaccharide (YesW/GalA-GalA) reveal that the enzyme adopts a β-propeller fold as a basic scaffold and has an active cleft at the center of the β-propeller (17Ochiai A. Itoh T. Maruyama Y. Kawamata A. Mikami B. Hashimoto W. Murata K. J. Biol. Chem. 2007; 282: 37134-37145Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), although the three-dimensional structure of YesX and the structural determinants for recognition of the substrate, especially Rha molecules, and the mode of action in PL family 11 RG lyases, are yet to be clarified. A synergistic catalysis by the endo- and exotype PLs plays an important role in initial degradation of the target polysaccharide. A plant-pathogenic Erwinia chrysanthemi 3937 secretes eight isoenzymes of pactate lyases such as PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ and one exotype polygalacturonate lyase PelX for degradation of polygalacturonan (18Shevchik V.E. Condemine G. Robert-Baudouy J. Hugouvieux-Cotte-Pattat N. J. Bacteriol. 1999; 181: 3912-3919Crossref PubMed Google Scholar). In alginate degradation by plant-related Sphingomoans sp. strain A1, three endotype alginate lyases, A1-I, A1-II, and A1-III, release oligosaccharides from the polymer, and the resultant oligoalginates are converted to the constituent monosaccharides through the reaction of exotype lyase, A1-IV (19Hashimoto W. Miyake O. Momma K. Kawai S. Murata K. J. Bacteriol. 2000; 182: 4572-4577Crossref PubMed Scopus (116) Google Scholar). Structure and function relationships in the endotype PLs have been demonstrated (2Yoder M.D. Jurnak F. Plant Physiol. 1995; 107: 349-364Crossref PubMed Scopus (61) Google Scholar, 3Mayans O. Scott M. Connerton I. Gravesen T. Benen J. Visser J. Pickersgill R. Jenkins J. Structure. 1997; 5: 677-689Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), but little information on the structural features of exotype lyases has been accumulated. Endotype YesW and exotype YesX from B. subtilis significantly resemble each other in primary structure, i.e. 68.7% identity in 597-amino acid overlap (16Ochiai A. Itoh T. Kawamata A. Hashimoto W. Murata K. Appl. Environ. Microbiol. 2007; 73: 3803-3813Crossref PubMed Scopus (61) Google Scholar), suggesting that their mode of action (endo/exo) is determined by a slight structural difference present in the catalytic domain. Structure comparison between YesW and YesX facilitates not only the identification of structural determinants for the mode of action but also the establishment of biotechnological bases of endo/exo interconversion in PLs. This article deals with the identification of structural determinants for substrate recognition and the mode of action in PL family 11 lyases through the determination of the crystal structures of YesW in complex with Rha (YesW/Rha) and YesX. On the basis of this structure and function relationship, exotype YesX was converted into YesW-like endotype enzyme by protein engineering. Materials-RG-I (from potatoes) was purchased from Megazyme. l-Rha was purchased from Wako Pure Chemical. Ni2+-chelating Sepharose™ Fast Flow and HiLoad™ 16/60 Superdex™ 200 pg were purchased from GE Healthcare. The RG chain, substrate for YesW and YesX, was prepared from RG-I as described previously (16Ochiai A. Itoh T. Kawamata A. Hashimoto W. Murata K. Appl. Environ. Microbiol. 2007; 73: 3803-3813Crossref PubMed Scopus (61) Google Scholar). Assays for Enzymes and Proteins-RG lyases was incubated at 30 °C for 5 min in a reaction mixture (1 ml) consisting of 0.5 mg/ml RG-I, 50 mm Tris-HCl (pH 7.5), and 2 mm CaCl2. Activity was determined by monitoring the increase in absorbance at 235 nm arising from the double bond formed in the reaction products. One unit (U) of enzyme activity was defined as the amount of enzyme required to produce an increase of 1.0 in absorbance at 235 nm/min using a cuvette with a light path 1 cm long. The protein content was determined by the Bradford method (20Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar), with bovine serum albumin as the standard. Protein Preparation-Protein expression and purification of YesW and YesX were conducted as described previously (16Ochiai A. Itoh T. Kawamata A. Hashimoto W. Murata K. Appl. Environ. Microbiol. 2007; 73: 3803-3813Crossref PubMed Scopus (61) Google Scholar, 21Ochiai A. Yamasaki M. Itoh T. Mikami B. Hashimoto W. Murata K. Acta Crystallogr. F Struct. Biol. Crystalliz. Comm. 2006; 62: 438-440Crossref PubMed Scopus (9) Google Scholar). Briefly, YesW and YesX expressed in Escherichia coli cells were purified through two-step column chromatography, i.e. Ni2+-chelating Sepharose™ Fast Flow and HiLoad™ 16/60 Superdex™ 200 pg. Each purified enzyme includes the C-terminal histidine-tagged sequence (eight amino acid residues, LEHHHHHH) derived from the expression vector pET21b (Novagen). The N-terminal 37 amino acid residues of YesW (total 628 amino acid residues) are excised as a signal peptide in E. coli cells (21Ochiai A. Yamasaki M. Itoh T. Mikami B. Hashimoto W. Murata K. Acta Crystallogr. F Struct. Biol. Crystalliz. Comm. 2006; 62: 438-440Crossref PubMed Scopus (9) Google Scholar). The molecular masses of the purified YesW and YesX including a C-terminal histidine tag were calculated to be 64,444 Da (591 amino acid residues) and 68,754 Da (620 amino acid residues), respectively. Crystallization and X-ray Diffraction-Crystallization of YesW was conducted as described previously (17Ochiai A. Itoh T. Maruyama Y. Kawamata A. Mikami B. Hashimoto W. Murata K. J. Biol. Chem. 2007; 282: 37134-37145Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 21Ochiai A. Yamasaki M. Itoh T. Mikami B. Hashimoto W. Murata K. Acta Crystallogr. F Struct. Biol. Crystalliz. Comm. 2006; 62: 438-440Crossref PubMed Scopus (9) Google Scholar). To analyze the complex form of YesW and Rha (YesW/Rha), a single crystal of YesW was soaked at 20 °C for 15 h in a solution containing 1.5 m Rha, 0.1 m Tris-HCl (pH 8.4), and 2 mm CaCl2. The crystal of YesX was prepared as follows. Purified YesX (7.5 mg/ml) in 20 mm Tris-HCl (pH 7.5) containing 2 mm CaCl2 and 0.2 m NaCl was crystallized at 20 °C by sitting drop vapor diffusion using Intelli-Plate (Veritas). The reservoir solution volume in each well was 0.1 ml, and the droplet was prepared by mixing 1 μl of the protein solution and 1 μl of the reservoir solution. A crystal suitable for x-ray analysis was obtained using a polyethylene glycol/ion screen kit (Hampton Research) for about a month. The reservoir solution for successful crystallization consisted of 20% polyethylene glycol 3350 and 0.2 m ammonium acetate. Crystals of YesW/Rha and YesX on a nylon loop (Hampton Research) were placed in a cold nitrogen gas stream at -173 °C, and x-ray diffraction images were collected at -173 °C under the nitrogen gas stream with a Jupiter 210 CCD detector and synchrotron radiation of wavelength 0.800 Å for the YesW/Rha crystal and 1.000 Å for the YesX crystal at the BL-38B1 station of SPring-8 (Hyogo, Japan). Additional cryoprotectant was required for vitrification of the YesX crystallization drop. To reduce the "ice rings," glycerol was added to the reservoir solution of the YesX crystal at a final concentration of 20%. Two hundred forty diffraction images from the crystal with 1.0° oscillation were collected as a series of consecutive data sets. Diffraction data were processed using the HKL2000 program package (22Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38617) Google Scholar). The data collection statistics are summarized in Table 1.TABLE 1Data collection and refinement statisticsYesW/RhaYesXSpace groupP21P212121Unit cell parameters (Å)a = 57.3, b = 105.9, c = 101.0, β = 94.8a = 72.9, b = 88.1, c = 99.3Data collectionWavelength (Å)0.8001.000Resolution limit (Å)50.0-1.32 (1.37-1.32)aThe data for the highest shells are given in parentheses.50.0-1.65 (1.71-1.65)Total reflections1,043,631747,425Unique reflections280,44077,151Redundancy3.8 (3.6)9.8 (9.1)Completeness (%)97.6 (95.1)98.9 (97.4)I/Sigma (I)13.0 (2.8)8.2 (4.1)Rmerge (%)6.6 (33.8)9.4 (25.8)RefinementFinal model1,164 (582 × 2) residues, 1,255 water molecules, 20 calcium ions,604 residues, 706 water molecules, 9 calcium ions,2 2-metyl-2,4-pentanediol, 7 rhamnose moleculesResolution limit (Å)50.0-1.32 (1.35-1.32)37.2-1.65 (1.70-1.65)Used reflections259,908 (18,169)72,365 (5,090)Completeness (%)97.4 (92.2)98.7 (94.9)Average B factor (Å2)Protein14.1, 15.3 (molecule A, B)9.6Water28.923.1Calcium ions11.87.72-Metyl-2,4-pentanediol34.1Rhamnose26.1R factor (%)16.7 (23.3)16.2 (19.8)Rfree (%)18.0 (25.8)18.4 (24.9)Root mean square deviationsBond (Å)0.0060.007Angle (°)1.171.10Ramachandran plot (%)Most favored regions89.389.3Additional allowed regions9.610.1Generously allowed regions1.00.6a The data for the highest shells are given in parentheses. Open table in a new tab Structure Determination and Refinement-The crystal structures of YesW/Rha and YesX were solved by molecular replacement using the Molrep program (23Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4175) Google Scholar) in the CCP4 program package (24Collaborative Computational ProjectActa Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19797) Google Scholar) with the ligand-free YesW structure (Protein Data Bank code 2Z8R) as a reference model. The Coot program (25Emsley P. Cowtan K. Acta Crystallogr. Sect. D. Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar) was used for manual modification of the initial model. Initial rigid body refinement, and several rounds of restrained refinement against the data set were done using the Refmac5 program (26Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar). Water molecules were incorporated where the difference in density exceeded 3.0 σ above the mean, and the 2Fo - Fc map showed a density of over 1.0 σ. At this stage, calcium ions were included in the calculation and refinement continued until convergence at maximum resolution (YesW/Rha, 1.32 Å; YesX, 1.65 Å). Protein models were superimposed, and their root mean square deviation was determined with the LSQKAB program (27Kabsch W. Acta Crystallogr. Sect. A. 1976; 32: 922-923Crossref Scopus (2380) Google Scholar), a part of CCP4. Final model quality was checked with PROCHECK (28Laskowski R.A. MacArthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). Ribbon plots were prepared using the PyMOL programs (29DeLano W.L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, San Carlos, CA2004Google Scholar). Coordinates used in this work were taken from the RCSB Protein Data Bank (30Berman H.M. Westbrook J. Feng Z. Gilliland G. Bhat T.N. Weissig H. Shindyalov I.N. Bourne P.E. Nucleic Acids Res. 2000; 28: 235-242Crossref PubMed Scopus (27935) Google Scholar). Deletion Mutagenesis-To delete the loop specific for YesX corresponding to 439PPGNDGMSY447, a YesX del_loop mutant was constructed using a KOD Plus Mutagenesis Kit (Toyobo). The plasmid, pET21b/YesX, constructed as described previously (16Ochiai A. Itoh T. Kawamata A. Hashimoto W. Murata K. Appl. Environ. Microbiol. 2007; 73: 3803-3813Crossref PubMed Scopus (61) Google Scholar), was used as a PCR template, and the following oligonucleotides were used as primers: sense, 5′-GGGCTTTTCACGAGCAAAGG-3′ and antisense, 5′-ATCAATTCCCCAGACGAGCGA-3′. PCR was performed according to the manufacturer's recommendation. Mutation was confirmed by DNA sequencing with an automated DNA sequencer (model 377; Applied Biosystems). Expression and purification of the mutants were conducted by the same procedures as those used for the wild-type YesX. Size Exclusion Chromatography-To determine the mode of action in the YesX del_loop mutant, the degradation profile of the RG chain through the YesW, YesX, or YesX del_loop mutant reaction was analyzed by size exclusion chromatography. Appropriate amounts of YesW, YesX, or YesX del_loop mutant were incubated at 30 °C for 60 min in a reaction mixture (1 ml) consisting of 0.5 mg/ml RG chain, 50 mm Tris-HCl (pH 7.5), and 2 mm CaCl2. The products were subjected to size exclusion chromatography using Superdex™ peptide 10/300 GL with AKTA purifier (GE Healthcare). Saccharides were eluted at a flow rate of 0.5 ml/min with 10 mm potassium phosphate (pH 7.0) and detected using a UV detector at 235 nm on the basis of C=C double bonds in the reaction products. Structure Determination of YesW/Rha-To identify YesW residues involved in substrate binding, we first tried to prepare the crystal of YesW in complex with the enzyme reaction product ΔGalA-Rha (16Ochiai A. Itoh T. Kawamata A. Hashimoto W. Murata K. Appl. Environ. Microbiol. 2007; 73: 3803-3813Crossref PubMed Scopus (61) Google Scholar) but failed. Thus, the crystal structure of YesW in complex with Rha (YesW/Rha) was determined at 1.32 Å resolution (Fig. 2). Data collection and refinement statistics are summarized in Table 1. The structure of YesW/Rha was solved by molecular replacement using the wild-type structure as a reference model. N-terminal amino acid residue and C-terminal histidine-tagged sequence (8 amino acid residues, -LEHHHHHH) could not be assigned in the 2Fo - Fc map. The refined model in an asymmetric unit consists of two identical monomers (582 amino acid residues × 2) termed molecules A and B. The root mean square deviation between molecules A and B was calculated as 0.293 Å for all residues (582 Cα atoms). On the basis of theoretical curves in the plot calculated according to Luzzati (31Luzzati V. Acta Crystallogr. 1952; 5: 802-810Crossref Google Scholar), the absolute positional error was estimated to be 0.13 Å at a resolution of 1.32 Å. Ramachandran plot analysis (32Sibanda B.L. Thornton J.M. Nature. 1985; 316: 170-174Crossref PubMed Scopus (510) Google Scholar), in which the stereochemical correctness of the backbone structure is indicated by (φ, ψ) torsion angles (33Ramachandran G.N. Sasisekharan V. Adv. Protein Chem. 1968; 23: 283-438Crossref PubMed Scopus (2770) Google Scholar), shows that 89.3% of nonglycine residues lie within the most favored regions and 9.6% of nonglycine residues lie in the additionally allowed regions. Five amino acid residues (Asn152, Ala327, Asn490, Ser506, and Ala594) in each molecule fell into generously allowed regions. One cispeptide was observed between Glu285 and Pro286 residues in each generously allowed region. Four Rha molecules in molecule A are well fitted in the electron density map (Fig. 3, A and C) with an average B factor of 23.4 Å2, whereas three Rha molecules were assigned in molecule B with an average B factor of 27.6 Å2. Because YesW is active in monomeric form (16Ochiai A. Itoh T. Kawamata A. Hashimoto W. Murata K. Appl. Environ. Microbiol. 2007; 73: 3803-3813Crossref PubMed Scopus (61) Google Scholar), interactions between the YesW molecule A and Rha molecules are focused on hereafter. The root mean square deviation between ligand-free YesW and YesW/Rha in molecule A was calculated as 0.129 Å for all residues (582 Cα atoms); this indicates that no significant conformational change occurs between protein structures with or without Rha molecules.FIGURE 3Rhamnose binding. A, electron density of Rha molecules in the active cleft by the omit map (Fo - Fc) calculated without Rha and countered at 3.0 σ. B, residues interacting with Rha molecules in the active cleft. C, electron density of Rha molecules in CBM-like domain by the omit map (Fo - Fc) calculated without the Rha and countered at 3.0 σ. D, residues interacting with Rha molecules in the CBM-like domain. Amino acid residues and Rha molecules are shown by colored elements: oxygen atom, red; carbon atom, blue in residues, yellow in Rha molecules RA1 and RA2, and cyan in RA3 and RA4; nitrogen atom, deep blue. The calcium ions are shown as orange balls, and water molecules are blue balls. Hydrogen bonds are shown as dashed lines. The characters indicate the saccharide number and its atoms in A and C and the amino acid residues number in B and D. E, structural superimposition in the active site between YesW/Rha and YesW/GalA-GalA. Rha and GalA-GalA molecules are shown by magenta and green sticks, respectively. The reaction product, ΔGalA-Rha, is fitted on the active site by structural simulation. ΔGalA is represented by light green sticks.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Rhamnose Binding-Two (RA1 and RA2) of four Rha molecules are bound to the active cleft, which is located at the center of β-propeller (Fig. 2A). Two (Tyr595 and Thr534) and four (Asn152, Asp172, Asn532, and Gly533) amino acid residues form a direct hydrogen bond (<3.4 Å) with RA1 and RA2, respectively (Fig. 3B and Table 2). In addition to direct hydrogen bonds, 10 water-mediated hydrogen bonds exist between the enzyme and Rha molecules: RA1/O-1=Wat951=Asn532/Nδ2 (2.9 Å); RA1/O-2=Wat658=Tyr595/OH (3.0 Å), =His214/N∈2 (3.3 Å), and =Asp178/Oδ1 (2.7 Å); RA2/O-1=Wat1097=Ser174/O (3.0 Å); RA2/O-2=Wat-951=Asn532/Nδ2 (2.8 Å); RA2/O-3= Wat852=Asp172/Oδ2 (2.9 Å); RA2/O-4=Wat711=Asn152/Oδ1 (3.0 Å), =Ala-151/O (2.7 Å), and =Ser150/Oγ (2.7 Å). van der Waals contacts (<4.5 Å) between RA1 and four amino acid residues (Tyr595, Lys535, Asn532, and Gly533) and RA2 and three amino acid residues (Gly533, Asn532, and Asn531) were observed. These amino acid residues are highly conserved in PL family 11 RG lyases (YesW and YesX from B. subtilis (16Ochiai A. Itoh T. Kawamata A. Hashimoto W. Murata K. Appl. Environ. Microbiol. 2007; 73: 3803-3813Crossref PubMed Scopus (61) Google Scholar), Rgl11A from Cellvibrio japonicus (34McKie V.A. Vincken J.P. Voragen A.G. van den Broek L.A. Stimson E. Gilbert H.J. Biochem. J. 2001; 355: 167-177Crossref PubMed Scopus (31) Google Scholar), and Rgl11Y from Clostridium cellulolyticum (35Pages S. Valette O. Abdou L. Belaich A. Belaich J.P. J. Bacteriol. 2003; 185: 4727-4733Crossref PubMed Scopus (44) Google Scholar)), indicating that these play important roles in recognizing the Rha residues of the RG chain.TABLE 2Interaction between YesW and RhaSugarHydrogen bond (<3.4 Å)C-C contact (<4.5 Å)AtomProteinAtomDistanceAtomProteinAtom(s)ÅRA1O-1Wat9512.6C-1Tyr595C∈ 1Wat6782.8C-2Tyr595C∈ 1, CζO-2Tyr595Oη3.0Lys535C∈, CγWat6583.0C-3Lys535C∈, CγWat6823.0Asn532CO-3Thr534Oγ12.8C-4Gly533CαO-4Thr534N3.0C-5Asn532CWat7972.7O-5Wat8252.7Wat6823.3RA2O-1Wat10972.6C-1Gly533CαWat11932.7Asn532CO-2Wat9513.0C-2Asn532CαO-3Asn152Oδ13.1C-3Asn532CαAsp172Oδ22.7C-4Asn532C, CαWat8523.1Asn531C, CαO-4Asn532N3.1C-5Gly533CαWat7112.7Asn532C, CαO-5Gly533N3.0RA3O-1Asp187Oδ12.7C-1Asp187CγOδ23.3C-2Lys207C∈Wat9183.3C-3Lys207C∈Wat8283.0C-4Arg255CζO-3Wat10152.9C-6Thr141Cα, CζWat10562.9Tyr147C∈ 2O-4Arg255Nη 12.8Gly186CαWat10563.2Wat7142.7O-5Wat10702.8RA4O-2Lys207Nζ2.9C-3Val246Cγ 1, Cγ 2O-3Gly238O2.6Gly238CαWat8282.8C-5Val246Cγ 2Wat8313.2C-6Lys240C∈O-4Wat8312.8 Open table in a new tab The other two Rha molecules (RA3 and RA4) are observed in the overhanging loop region formed in the blade C of the β-propeller domain (Fig. 2). Two amino acid residues (Asp187 and Arg255) form a direct hydrogen bond with RA3 and two (Lys207 and Gly238) with RA4 (Fig. 3D, Table 2). The 19 water-mediated hydrogen bonds also exist between the enzyme and Rha molecules: RA3/O-1=Wat918=Asp187/Oδ2 (2.7 Å); RA3/O-1=Wat828=Lys207/Nζ (2.8 Å) and =Lys207/N (2.9 Å); RA3/O-3=Wat1015=Lys207/Nζ (3.0 Å); RA3/O-3=Wat1056=Arg255/Nη2 (2.9 Å); RA3/O-4=Wat1056=Arg255/Nη2 (2.9 Å); RA3/O-4=Wat714=Thr140/O (2.8 Å) and =Pro142/N (3.1 Å); RA3/O5=Wat1070=Asp-187/Oδ2 (3.2 Å), =Asp187/N (2.8 Å), and =Tyr147/OH (2.4 Å); RA4/O-3=Wat828=Lys207/Nζ (2.8 Å) and =Lys207/N (2.9 Å); RA4/O-3=Wat831=Gly238/O (2.9 Å), =Asn204/O (3.1 Å), and =Asn204/Oδ1 (3.2 Å); RA4/O-4=Wat831=Gly238/O (2.9 Å), =Asn204/O (3.1 Å), and =Asn204/Oδ1 (3.2 Å). van der Waals contacts between RA3 and six amino acid residues (Asp187, Lys207, Arg255, Thr141, Tyr147, and Gly186) and between RA4 and three amino acid residues (Val246, Gly238, and Lys240) were observed. A space between RA3 and RA4 suggests that GalA, which forms a α-1,4 bond with RA3 and α-1,2 bond with RA4, is accommodated at this site. In this case, the basic residues such as Arg255 and/or Lys207 are supposed to stabilize the carboxyl group of the GalA residue. Rha molecules are unexpectedly bound to the noncatalytic domain in addition to the active cleft. This noncatalytic domain for Rha binding probably function as a carbohydrate-binding module (CBM). Some carbohydrate-active enzymes are appended by one or more noncatalytic CBMs, which are classified into 52 families based on their amino acid sequence similarity and promote association of the enzyme and substrate (36Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Biochem. J. 2004; 382: 769-781Crossref PubMed Scopus (1537) Google Scholar). A CBM family 32 protein, YeCBM32 from Yersinia enterolitica, has been reported to recognize a
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