Journal of Molecular Biology
Volume 377, Issue 5, 11 April 2008, Pages 1443-1459
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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

https://doi.org/10.1016/j.jmb.2008.01.090Get rights and content

Abstract

The three-dimensional structure of a Salmonella enterica hypothetical protein YihS is significantly similar to that of N-acyl-d-glucosamine 2-epimerase (AGE) with respect to a common scaffold, an α66-barrel, although the function of YihS remains to be clarified. To identify the function of YihS, Escherichia coli and S. enterica YihS proteins were overexpressed in E. coli, purified, and characterized. Both proteins were found to show no AGE activity but showed cofactor-independent aldose–ketose isomerase activity involved in the interconversion of monosaccharides, mannose, fructose, and glucose, or lyxose and xylulose. In order to clarify the structure/function relationship of YihS, we determined the crystal structure of S. enterica YihS mutant (H248A) in complex with a substrate (d-mannose) at 1.6 Å resolution. This enzyme–substrate complex structure is the first demonstration in the AGE structural family, and it enables us to identify active-site residues and postulate a reaction mechanism for YihS. The substrate, β-d-mannose, fits well in the active site and is specifically recognized by the enzyme. The substrate-binding site of YihS for the mannose C1 and O5 atoms is architecturally similar to those of mutarotases, suggesting that YihS adopts the pyranose ring-opening process by His383 and acidifies the C2 position, forming an aldehyde at the C1 position. In the isomerization step, His248 functions as a base catalyst responsible for transferring the proton from the C2 to C1 positions through a cis-enediol intermediate. On the other hand, in AGE, His248 is thought to abstract and re-adduct the proton at the C2 position of the substrate. These findings provide not only molecular insights into the YihS reaction mechanism but also useful information for the molecular design of novel carbohydrate-active enzymes with the common scaffold, α66-barrel.

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 α66-barrel structure with a deep pocket. In contrast to the abundance of α-helices, there is little β-sheet structure (Fig. 1a). The α66-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 (α66-barrel),1, 2, 12, 13, 14, 15, 16, 17 seven-hairpin glycosidases (α77-barrel),18 family PL5 polysaccharide lyases (α65-barrel),19 family PL8 polysaccharide lyases (α55-barrel),20, 21, 22, 23 family PL10 polysaccharide lyases (α33-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 α66-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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