Journal of Molecular Biology
Crystal Structure of YihS in Complex with d-Mannose: Structural Annotation of Escherichia coli and Salmonella enterica yihS-encoded Proteins to an Aldose–Ketose Isomerase
Introduction
Increasingly, for numerous DNA sequences and crystal structures, structural genomics provides valuable information on the intrinsic function of hypothetical proteins. Through structural genomic approaches, we have identified Bacillus subtilis hypothetical proteins YteR and YesR, whose crystal structures are very similar to those of unsaturated glucuronyl hydrolases (family GH88),1 to be novel carbohydrate-metabolizing enzymes, unsaturated galacturonyl hydrolases (family GH105).2 Furthermore, we have proved that their catalytic mechanisms are comparable.3, 4 Similarly, based on the three-dimensional structure in the Protein Data Bank (PDB), Salmonella enterica hypothetical protein YihS (SeYihS) exhibits a high degree of similarity with N-acyl-d-glucosamine 2-epimerases (AGEs), although little amino acid sequence identity is observed between the two, as is the case in family GH88 and GH105 enzymes. The crystal structure of SeYihS was first determined as that of a hypothetical protein of unknown function by the New York Structural Genomics Research Consortium (NYSGRC). Detailed analysis of the SeYihS structure has not, to the best of our knowledge, been published, although its coordinates are available in the PDB (2AFA, deposited on July 25, 2005). Proteins encoded by yihS genes have been defined as AGEs, based on this structural similarity (Pfam, GlcNAc 2-epimerase family, accession no. PF07221).5 On the other hand, AGE catalyzes the reversible epimerization between N-acetyl-d-glucosamine (GlcNAc) and N-acetyl-d-mannosamine (ManNAc).6 The physiological significance of AGE in the formation of N-acetyl neuraminic acid in mammals remains unclear, although the biosynthesis of N-acetyl neuraminic acid has been studied extensively in vivo and in vitro.7 AGE was isolated from porcine kidney tissue and characterized enzymatically.8, 9 We previously determined the crystal structure of porcine AGE (PDB code 1FP3, deposited on August 30, 2000) in order to clarify the physiological role of AGE.10 Moreover, the crystal structure of Anabaena sp. CH1 AGE (Anabaena AGE) was recently determined by Lee et al.11
A subunit of YihS and AGE is composed of 12 α-helices constituting an α6/α6-barrel structure with a deep pocket. In contrast to the abundance of α-helices, there is little β-sheet structure (Fig. 1a). The α6/α6-barrel structure consists of 6 outer helices running in roughly the same direction and 6 inner helices oriented in the opposite direction. These helices are connected in a nearest-neighbor and an up-and-down pattern by short and long loops. This α/α-barrel family is shared by six-hairpin glycosidases (α6/α6-barrel),1, 2, 12, 13, 14, 15, 16, 17 seven-hairpin glycosidases (α7/α7-barrel),18 family PL5 polysaccharide lyases (α6/α5-barrel),19 family PL8 polysaccharide lyases (α5/α5-barrel),20, 21, 22, 23 family PL10 polysaccharide lyases (α3/α3-barrel),24 and terpenoid cyclases/protein prenyltransferases25 in the SCOP† database.26 Almost α/α-barrel structures were found in carbohydrate-active enzymes. Clarification of their evolutionary relationships has been far more difficult. We cannot determine whether the disparate α/α-barrel enzymes could evolve from a single protein ancestor or evolved convergently from different protein ancestors. However, their common architecture of the binding pocket is thought to be suitable for carbohydrate-active enzymes. This suggests that with this basic scaffold, we can develop novel enzymes that have novel substrate specificity and reactions.
Here, we describe the overexpression and characterization of Escherichia coli- and S. enterica yihS-encoded proteins. The enzymatic properties indicate that YihS shows no AGE activity, but does show enzyme activity, that of aldose–ketose isomerase, with monosaccharides. This result completely rules out the previously accepted definition of YihS as an AGE. Furthermore, in this study, we discuss in detail the crystal structure of a mutant YihS of S. enterica in complex with a substrate (d-mannose), and we determine the active-site residues responsible for substrate binding and enzyme catalysis. The data obtained in this study demonstrate the intrinsic function of YihS, provide molecular insights into the catalytic reaction mechanism, and propose a novel enzyme-design strategy with the α6/α6-barrel basic scaffold.
Section snippets
Structure-based comparison of YihS and AGE
The three-dimensional structures of SeYihS (PDB code 2AFA, deposited on July 25, 2005) and porcine AGE (PDB code 1FP3, deposited on August 30, 2000) show the highest degree of similarity in the PDB (Fig. 1). The crystal structure of SeYihS was first determined as a hypothetical protein of unknown function by the NYSGRC. YihS proteins are widely distributed in bacteria, e.g., Escherichia, Salmonella, Shigella, Vibrio, Arthrobacter, and Pseudomonas (Supplementary Fig. S1). BLAST27 and ClustalW28
Discussion
In this study, we identified yihS-encoded proteins, which are broadly present in bacteria, as aldose–ketose isomerases catalyzing the reversible conversion of Man, Fru, and Glc, or Lyx and Xul (Fig. 2 and Table 1), although YihS has previously been designated as an AGE.
Mannose isomerases catalyzing Man and Fru isomerization have been studied in several bacterial species—Pseudomonas,37 Mycobacterium,38 Escherichia,39, 40 and Agrobacterium41—and commercially utilized to produce Man, although
Chemicals and reagents
All chemicals and reagents were of analytical grade and purchased from Wako Pure Chemical (Osaka, Japan) or Sigma (St. Louis, MO), unless otherwise noted.
Molecular cloning of E. coli and S. enterica YihS genes
Gene cloning was carried out according to the standard method.57 To subclone the EcYihS gene into an expression vector, pET21b (Novagen, Madison, WI), a colony direct PCR was performed using KOD plus polymerase (Toyobo, Tokyo, Japan), a single colony of E. coli K-12 (MG1655) as a template, and two synthetic oligonucleotides (Hokkaido System
Acknowledgements
We thank Drs. K. Hasegawa and H. Sakai of the Japan Synchrotron Radiation Research Institute (JASRI) for their valuable help in data collection. Diffraction data sets for the crystals were collected from the BL-38B1 station of SPring-8 with approval from the JASRI. Computation time was provided by the Supercomputer Laboratory Institute for Chemical Research, Kyoto University, Japan. This work was supported in part by Grants-in-Aid for Scientific Research, COE for Microbial-Process Development
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