Journal of Molecular Biology
Volume 354, Issue 4, 9 December 2005, Pages 927-939
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The Structure of 3-Deoxy-d-arabino-heptulosonate 7-phosphate Synthase from Mycobacterium tuberculosis Reveals a Common Catalytic Scaffold and Ancestry for Type I and Type II Enzymes

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The shikimate pathway, responsible for the biosynthesis of aromatic compounds, is essential for the growth of Mycobacterium tuberculosis and is a potential target for the design of new anti-tuberculosis drugs. The first step of this pathway is catalyzed by 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS). The DAH7PSs have been classified into two apparently unrelated types and, whereas structural data have been obtained for the type I DAH7PSs, no structural information is available for their type II counterparts. The type II DAH7PS from M. tuberculosis has been expressed in Escherichia coli, purified, functionally characterized and crystallized. It is found to be metal ion-dependent and subject to feedback inhibition by phenylalanine, tryptophan, tyrosine and chorismate, with a significant synergistic effect when tryptophan is used in combination with phenylalanine. The crystal structure of M. tuberculosis DAH7PS has been determined by single-wavelength anomalous diffraction and refined at 2.3 Å in complex with substrate phosphoenolpyruvate and Mn2+. The structure reveals a tightly associated dimer of (β/α)8 TIM barrels. The monomer fold, the arrangement of key residues in the active site, and the binding modes of PEP and Mn2+, all match those of the type I enzymes, and indicate a common ancestry for the type I and type II DAH7PSs, despite their minimal sequence identity. In contrast, the structural elements that decorate the core (β/α)8 fold differ from those in the type I enzymes, consistent with their different regulatory and oligomeric properties.

Introduction

Tuberculosis (TB) is an ancient disease but remains one of the most serious health threats facing mankind. Worldwide deaths total 2–3 million annually,1 more than for any other infectious disease, and it is estimated that approximately one-third of the world's population is infected. Although effective anti-TB drugs exist, the long treatment times required, the problems of latent or persistent TB,2 and the proliferation of multidrug-resistant strains of Mycobacterium tuberculosis, the causative agent,3 have all created an urgent need for the development of new antimycobacterial agents. This is one of the most important of current challenges for drug discovery.

The shikimate pathway comprises a series of seven enzyme-catalyzed reactions that result in the biosynthesis of chorismate, which is the precursor for many essential aromatic compounds.4 These include the aromatic amino acids tryptophan, tyrosine and phenylalanine; folic acid, an essential cofactor for many enzymatic processes; and salicylate, used for the biosynthesis of the siderophores through which bacteria acquire iron.5 The shikimate pathway is found in microorganisms and plants, and was discovered recently in the apicomplexan parasites Plasmodium falciparum, Toxoplasma gondii, and Cryptosporidium parvum.6, 7 However, the pathway is absent from higher organisms, making the enzymes of this pathway attractive as targets for the development of antibiotics and herbicides. The active ingredient of the herbicide Roundup®, glyphosate [N-(phosphomethyl)glycine], which inhibits the sixth enzyme of the shikimate pathway,8 5-enolpyruvyl shikimate-3-phosphate synthase, has been shown to inhibit the growth of apicomplexan parasites in vitro.6 Recent gene disruption studies have shown that operation of the shikimate pathway is essential for the viability of M. tuberculosis,9 validating the choice of enzymes from this pathway as targets for the development of novel antimycobacterial agents.

The first committed step in the shikimate pathway is catalyzed by the enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS, EC 2.5.1.54). This enzyme catalyzes an aldol-like condensation reaction between phosphoenolpyruvate (PEP) and d-erythrose 4-phosphate (E4P) to generate 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAH7P) and inorganic phosphate. DAH7PSs have been classified into two, apparently unrelated, types.10 Type I DAH7PSs are smaller than their type II counterparts, with molecular masses less than 40 kDa, and can be further divided into two sequence subfamilies, Iα and Iβ.11, 12 The type Iα and Iβ DAH7PSs have been well characterized, structurally and functionally. They share a divalent metal ion dependence and susceptibility to feedback inhibition, and crystal structures of the type Iα enzymes from Escherichia coli13, 14, 15 and Saccharomyces cerevisiae,16, 17 and the type Iβ enzymes from Thermotoga maritima18 and Pyrococcus furiosus,19 reveal a common (β/α)8 TIM barrel fold.

Analysis of the genome of M. tuberculosis reveals the presence of a single open reading frame (Rv2178c) encoding a putative DAH7PS of 462 residues.20 This belongs to the type II DAH7PS family, a separate homologous group of enzymes, distinct in size (∼50 kDa) and sequence from the type I enzymes.11, 21 Type II enzymes were first identified in plants,22, 23 and subsequently a limited number of microbial examples were characterized.10 As more microbial genomes have been sequenced, however, the number of type II DAH7PSs known has grown rapidly, and it appears that this type of DAH7PS consists of a subset of plant enzymes clustered within a more divergent set of microbial enzymes.21 In some organisms, genes encoding both type I and type II DAH7PSs have been identified, with several of the type II enzymes apparently being required for the biosynthesis of specific secondary metabolites.24, 25 However, the presence of only type II DAH7PSs in the predicted proteomes of a number of species including Streptomyces sp., Corynebacterium diphtheriae, Campylobacter jejuni, Agrobacterium tumefaciens, Novosphingobium aromaticivorans, Helicobacter pylori and several Mycobacteria species provides evidence for a role of type II DAH7PSs in aromatic amino acid biosynthesis.

Sequence identity between the type I and type II DAH7PSs is less than 10%, raising the possibility that they represent distinct protein families, unrelated by evolution. The third enzyme of the shikimate pathway, dehydroquinase, an enzyme that functions also in quinic acid metabolism, is similarly found in two distinct protein families. The two enzyme types exhibit altered catalytic mechanism and are distinctly different in structure, indicating that despite common overall reaction chemistry, the two groups arose via convergent evolution.26, 27 Recent biochemical characterization of the type II enzyme from H. pylori has indicated many key mechanistic features that are shared between type I and type II DAH7PSs,28 but no three-dimensional structural information is available for any type II enzyme. However, the presence of type II DAH7PSs in a number of important pathogenic bacteria makes them prime targets for study. DAH7PS is a key drug target in its own right, given its crucial role in the biosynthesis of many key aromatic molecules, and the exploitation of differences between the two types of DAH7PS may enable the development of narrow-spectrum antibiotics.

Here, we report the functional characterization and three-dimensional structure of the type II DAH7PS from M. tuberculosis (Mt-DAH7PS). The crystal structure reveals a striking correspondence of both fold and function in the two types of DAH7PS, despite the minimal sequence identity, with a common catalytic apparatus on a shared (β/α)8 TIM barrel fold. Importantly, however, distinct decorations of the basic fold and modes of oligomerization suggest different regulation of the two types and illuminate the divergent evolutionary relationships between these two families.

Section snippets

Expression and functional characterization

The expression of Mt-DAH7PS in E. coli gave rise to insoluble protein, and no enzymic activity was detected in the supernatant following cell lysis and centrifugation. On the basis of our recent observation that H. pylori type II DAH7P synthase is a substrate for E. coli chaperonins, we coexpressed GroEL/GroES and Mt-DAH7PS, obtaining soluble (and active) recombinant protein.28 Size-exclusion chromatography gave a molecular mass of ∼100 kDa, consistent with a dimeric species. The apparent KM values

Discussion

The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) is essential for the viability of most organisms because of its key role in chorismate biosynthesis. Chorismate, in turn, is the precursor for a wide variety of essential aromatic compounds, some of them universally required, such as folic acid and the aromatic amino acids, and others restricted to particular species, such as phenazine and the salicylate-based siderophores. Several subtypes of DAH7PS are often found in a

Conclusions

These studies on the type II DAH7PS from M. tuberculosis reveal a remarkable similarity in fold and function between type I and type II enzymes. Despite their minimal sequence identity, and the significant differences in quaternary structure and extensions to the core (β/α)8 barrel, the key residues that interact with PEP and the divalent metal ion are completely conserved and positioned almost identically in the two DAH7PS types. Recent studies with the type II DAH7PS from H. pylori show that

Protein expression and purification

The predicted opening reading frame encoding Mt-DAH7PS (Rv2178c) was amplified from M. tuberculosis H37Rv genomic DNA using the polymerase chain reaction (PCR). The PCR product was cloned into pProExHTa (Novagen) expression vector, yielding a protein containing an N-terminal tobacco etch virus (TEV) protease-cleavable His6 tag upstream of the predicted translation start site. For the production of soluble protein, the plasmid was transformed into E. coli BL21 (DE3) cells containing the pGroESL

Acknowledgements

We thank Mark Patchett for the pGroESL plasmid, Gillian Norris for assistance with crystal mounting, and Minmin Yu for synchrotron data collection at the Advanced Light Source (Berkeley, CA). The assistance of Geoffrey Jameson and Bryan Anderson with the structure refinement, and Richard Bunker with the preparation of Figures, is gratefully acknowledged. C.J.W. is a recipient of a Massey University Vice Chancellor's Doctoral Scholarship. This work was funded by the Royal Society of New Zealand

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