Crystal structure of N-acyl-d-glucosamine 2-epimerase from porcine kidney at 2.0 Å resolution

Itoh, Takafumi; Mikami, Bunzo; Maru, Isafumi; Ohta, Yasuhiro; Hashimoto, Wataru; Murata, Kousaku

doi:10.1006/jmbi.2000.4188

Cited by 60 publications

(61 citation statements)

References 33 publications

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“…Glucoamylase is a polysaccharide exo-hydrolase found in some prokaryotic and many eukaryotic microorganisms. AGE has the ␣ 6 /␣ 6 -barrel structure we determined, and catalyzes the epimeric reaction for N-acetyl-D-glucosamine and N-acetyl-D-mannosamine (41). Fig.…”

Section: Resultsmentioning

confidence: 94%

“…Based on the structural similarity in the RCSB Protein Data Bank (28) observed with the DALI (29) program, three proteins, i.e. hypothetical protein Yter from B. subtilis, glucoamylase from Thermoanaerobacterium thermosaccharolyticum (36), and AGE from the porcine kidney (41), in the SCOP data base superfamily exhibited the highest degree of similarity. These proteins exhibited Z-scores of 32.7, 21.1, and 19.4.…”

Section: Resultsmentioning

confidence: 99%

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Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

Itoh

Akao

Hashimoto

et al. 2004

Journal of Biological Chemistry

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Unsaturated glucuronyl hydrolase (UGL) is a novel glycosaminoglycan hydrolase that releases unsaturated D-glucuronic acid from oligosaccharides produced by polysaccharide lyases. The x-ray crystallographic structure of UGL from Bacillus sp. GL1 was first determined by multiple isomorphous replacement (mir) and refined at 1.8 Å resolution with a final R-factor of 16.8% for 25 to 1.8 Å resolution data. The refined UGL structure consists of 377 amino acid residues and 478 water molecules, four glycine molecules, two dithiothreitol (DTT) molecules, and one 2-methyl-2,4-pentanediol (MPD) molecule. UGL includes an ␣ 6 /␣ 6 -barrel, whose structure is found in the six-hairpin enzyme superfamily of an ␣/␣-toroidal fold. One side of the UGL ␣ 6 /␣ 6 -barrel structure consists of long loops containing three short ␤-sheets and contributes to the formation of a deep pocket. One glycine molecule and two DTT molecules surrounded by highly conserved amino acid residues in UGLs were found in the pocket, suggesting that catalytic and substrate-binding sites are located in this pocket. The overall UGL structure, with the exception of some loops, very much resembled that of the Bacillus subtilis hypothetical protein Yter, whose function is unknown and which exhibits little amino acid sequence identity with UGL. In the active pocket, residues possibly involved in substrate recognition and catalysis by UGL are conserved in UGLs and Yter. The most likely candidate catalytic residues for glycosyl hydrolysis are Asp 88 and Asp 149 . This was supported by site-directed mutagenesis studies in Asp 88 and Asp 149 .

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Section: Resultsmentioning

confidence: 94%

Section: Resultsmentioning

confidence: 99%

Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

Itoh

Akao

Hashimoto

et al. 2004

Journal of Biological Chemistry

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“…Structure-based alignment of the amino acid sequence of RaCE (accession no. BAF81108) was done with those of N-acetyl-D-glucosamine 2-epimerase (AGE) from porcine kidney (pAGE; PDB code 1fp3) (Itoh et al 2000) and that from Anabaena sp. CH1 (aAGE; PDB code 2gz6) (Lee et al 2007).…”

Section: Structure-based Alignment and Homology Modeling Of Racementioning

confidence: 99%

“…Mutants with R52A, R52K, R52H, H243A, H243R, E246A, E246Q, E308A, E308D, E308Q, H374A, H374K, and H374R completely lost the activity toward cellobiose and lactose, indicating that R52, H243, E246, E308 and H374 are absolutely required for catalysis. Itoh et al (2000) suggested that R60 (R52), H248 (H243), E251 (E246), and H372 (H374) played an important role in the reaction of pAGE base on X-ray crystallographic analysis, and Lee et al (2007) identified R57 (R52), H239 (H243), E308 (E308), and H378 (H374) as critical residues and E242 (E246) and R375 (R377) as essential residues in aAGE by site-directed mutagenesis (the numbers in parentheses indicate the corresponding residues in RaCE). Mutant with R377H exhibited no activity but one with R377A retained 43% of wild-type enzyme toward cellobiose and 23% toward lactose, suggesting that the conserved R377 may not be essential for catalysis in RaCE.…”

Section: Mutation Of Ionizable Residuesmentioning

confidence: 99%

Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus

et al. 2009

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sp. CH1 and on the computer-aided model building of the tertiary structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. It was found that R52, H243, E246, W249, M251, W304, E308, H374, and M378 were absolutely required for the activity of RaCE. F114 and W303 (and possibly R377) also contributed to catalysis. These possible catalytic residues protruded into or near the active-site cleft surrounded by the inner -helices in the predicted (/) 6 core barrel structure of RaCE.

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“…53) As for the = -barrel, we have confirmed that a porcine kidney bifunctional protein, renin-binding protein [N-acyl-D-glucosamine 2-epimerase (AGE)], 54,55) a novel glycosaminoglycan-degrading enzyme of unsaturated glucuronyl hydrolase (UGL) of Bacillus sp. GL1, 56,57) a plant cell wall-degrading enzyme (B. subtilis unsaturated galacturonyl hydrolase) YteR, 58) and an AGE structural homolog, YihS, (unpublished) of S. typhimurium have = -barrel structures as a basic scaffold.…”

Section: Structural Features Of Polysaccharide Lyasementioning

confidence: 80%

Superchannel of Bacteria: Biological Significance and New Horizons

Murata

Kawai

Mikami

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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Crystal structure of N-acyl-d-glucosamine 2-epimerase from porcine kidney at 2.0 Å resolution

Cited by 60 publications

References 33 publications

Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

Crystal Structure of Unsaturated Glucuronyl Hydrolase, Responsible for the Degradation of Glycosaminoglycan, from Bacillus sp. GL1 at 1.8 Å Resolution

Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus

Superchannel of Bacteria: Biological Significance and New Horizons

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