Journal of Molecular Biology
Volume 406, Issue 2, 18 February 2011, Pages 325-342
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Structural Studies of ROK Fructokinase YdhR from Bacillus subtilis: Insights into Substrate Binding and Fructose Specificity

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Abstract

The main pathway of bacterial sugar phosphorylation utilizes specific phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In addition to the classic PTS system, a PTS-independent secondary system has been described in which nucleotide-dependent sugar kinases are used for monosaccharide phosphorylation. Fructokinase (FK), which phosphorylates d-fructose with ATP as a cofactor, has been shown to be a member of this secondary system. Bioinformatic analysis has shown that FK is a member of the “ROK” (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) sequence family. In this study, we report the crystal structures of ROK FK from Bacillus subtilis (YdhR) (a) apo and in the presence of (b) ADP and (c) ADP/d-fructose. All structures show that YdhR is a homodimer with a monomer composed of two similar α/β domains forming a large cleft between domains that bind ADP and d-fructose. Enzymatic activity assays support YdhR function as an ATP-dependent fructose kinase.

Graphical Abstract

Introduction

Phosphorylation of monosaccharides is a critical step in carbohydrate metabolism. This fundamental reaction traps sugars inside the cell and targets them for further utilization by specific metabolic pathways. The main pathway of sugar phosphorylation in bacteria utilizes phosphoenolpyruvate phosphotransferase system (PTS) enzymes. In general, the bacterial PTS system has two cytoplasmic proteins: a phosphocarrier protein and phosphotransferase and a membrane-bound sugar-specific transporter.1, 2, 3 The transporter can be part of a large multidomain protein system or could function as a specific permease. In addition to the PTS system, a PTS-independent secondary system has been described for a number of bacteria.4 These systems utilize nucleotide-dependent sugar kinases capable of phosphorylating monosaccharides. Many bacterial genomes contain genes for hexokinases, glucokinases, or fructokinases (FKs) capable of phosphorylating a wide variety of sugar substrates. Hexokinases are known to catalyze the phosphorylation of a broader range of monosaccharides such as glucose, mannose, and fructose, while they are present in high concentrations.5 In contrast, glucokinases and FKs are much more selective with a relatively high affinity for glucose and fructose, respectively.

Bioinformatic and structural studies of 60 different sugar kinases have revealed three distinct families of kinases: the galactokinase, hexokinase, and ribokinase families.4 In addition, Titgemayer and co-workers have noticed that some sugar kinases share a close amino acid sequence relationship to proteins with divergent (or unknown) functions and proposed that they belong to a novel family of proteins called the “ROK family.”6 On the basis of the amino acid sequence similarity, several sugar kinases and transcriptional repressors and a large number of uncharacterized proteins have been grouped into the ROK (bacterial Repressors, uncharacterized Open reading frames, and sugar Kinases) family of enzymes. Currently, there are nearly 5000 proteins annotated (Pfam PF00480)7 as members of the ROK family. The members of this family are broadly distributed in nature and are found in bacteria, archaea, eukaryota, plants, and humans. Also, many ROK sugar kinases phosphorylate a wide range of carbohydrate substrates showing high functional diversity within the family.6, 8

To date, only few members of this protein family have been structurally characterized; hence, structural coverage of this large and important family of proteins is very limited. The well-known members of this family are the xylose operon repressor (XylR),9, 10 N-acetyl-glucosamine repressor (NagC), glucokinase (EC: 2.7.1.2),11 allokinase (EC: 2.7.1.55),12 ATP-glucomannokinase (GMK),13 and Mlc repressor.14 In order to better structurally characterize this large, important, and functionally diverse ROK family of proteins and to understand structure/function relationships, the Midwest Center for Structural Genomics (MCSG), a component of the Protein Structure Initiative (PSI), initiated the structural studies of several representatives of this family. Here, we present the first crystal structure of ROK fructokinase (YdhR) from Bacillus subtilis. We have determined the 2.10-Å apo structure (YdhR-apo) and 1.66-Å structure of the enzyme with ADP bound (YdhR-ADP) and a 2.45-Å structure with ADP and d-fructose (YdhR-ADP-Fru) molecules bound. The YdhR monomer is composed of two similar α/β domains and assembles into a homodimer that is observed in solution and in the crystal. Our structures revealed that the YdhR fold closely resembles that of the actin-like ATPase domain superfamily (CATH 3.30.420.160). The active site is located in a large cleft between domains of the monomer with ADP and d-fructose bound in the middle of the cleft. Fructokinase activity was confirmed by enzymatic assay. Also, point mutation of glycine 59 to alanine showed that this residue plays a critical role in the specificity of YdhR to d-fructose. A single zinc ion was found bound to the protein near the active site, where it orients active-site His153.

Section snippets

Enzymatic activity

The B. subtilis ROK FK protein (YdhR, Swiss-Prot entry O05510) was predicted to have putative fructokinase function based on genome sequence analysis.15, 16 Searches of the Kyoto Encyclopedia of Genes and Genomes (KeGG) database indicated that YdhR might be involved in fructose and mannose metabolism as well as starch and sucrose metabolism (Pathways bsu00051, bsu00500).17 Our initial YdhR structural analysis revealed a phosphotransferase-like protein. In order to confirm whether this enzyme is

Conclusions

Here, we present the first structural and functional analysis of YdhR fructokinase, a member of the ROK family. We have determined the structure of an apo, ADP-bound, and ADP-d-fructose-bound YdhR enzyme and confirmed its fructokinase function. This is only the second extensive structural study of an enzyme in this large and versatile family and only the second structure of an ROK enzyme solved in the presence of its substrate. Superimposition of three structures of YdhR does not reveal any

Gene cloning and protein expression

The open reading frame of YdhR (gi|3914959) was amplified by PCR from B. subtilis MC58 genomic DNA (ATCC) with KOD DNA polymerase, using the conditions and reagents provided by the vendor (Novagen, Madison, WI). The gene was cloned into the pMCSG7 vector by using a modified ligation-independent cloning protocol.43 This process generated an expression clone of a fusion protein with an N-terminal His6-tag and a TEV protease recognition site (ENLYFQ↓S). The fusion protein was expressed in an E.

Acknowledgements

We wish to thank all the members of the Structural Biology Center at Argonne National Laboratory for their help in conducting these experiments. This work was supported by National Institutes of Health grant GM074942 and by the U.S. Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357. The submitted manuscript has been created by UChicago Argonne, LLC, Operator of Argonne National Laboratory (“Argonne”). Argonne, a U.S. Department of Energy

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    The submitted manuscript has been created by UChicago Argonne, LLC, Operator of Argonne National Laboratory (“Argonne”). Argonne, a U.S. Department of Energy Office of Science laboratory, is operated under Contract No. DE-AC02-06CH11357. The U.S. Government retains for itself, and others acting on its behalf, a paid-up nonexclusive, irrevocable worldwide license in said article to reproduce, prepare derivative works, distribute copies to the public, and perform publicly and display publicly, by or on behalf of the Government.

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