Journal of Molecular Biology
Volume 364, Issue 3, 1 December 2006, Pages 411-423
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Mechanism of Phosphoryl Transfer Catalyzed by Shikimate Kinase from Mycobacterium tuberculosis

https://doi.org/10.1016/j.jmb.2006.09.001Get rights and content

Abstract

The structural mechanism of the catalytic functioning of shikimate kinase from Mycobacterium tuberculosis was investigated on the basis of a series of high-resolution crystal structures corresponding to individual steps in the enzymatic reaction. The catalytic turnover of shikimate and ATP into the products shikimate-3-phosphate and ADP, followed by release of ADP, was studied in the crystalline environment. Based on a comparison of the structural states before initiation of the reaction and immediately after the catalytic step, we derived a structural model of the transition state that suggests that phosphoryl transfer proceeds with inversion by an in-line associative mechanism. The random sequential binding of shikimate and nucleotides is associated with domain movements. We identified a synergic mechanism by which binding of the first substrate may enhance the affinity for the second substrate.

Introduction

The shikimate pathway converts erythrose-4-phosphate into chorismate, which is an intermediate required for the biosynthesis of aromatic amino acids and other important aromatic metabolites. The pathway is used in algae, higher plants, bacteria, fungi, and apicomplexan.1., 2. It is likely to have been an ancient eukaryotic attribute, which has been lost in many taxonomes that are now dependent on exogenous aromatic compounds.2 In particular, the pathway is absent from mammals,3 which makes enzymes in the pathway potential targets for the development of non-toxic antimicrobial agents, herbicides, and anti-parasitic drugs.

The genomic organization of the aro genes in the shikimate pathway of Mycobacterium tuberculosis includes one operon, aroCKBQ, and three isolated aro genes, aroE, aroG and aroA.4 The aroK gene is essential in M. tuberculosis.4 It encodes the enzyme shikimate kinase (SK, EC 2.7.1.71), which catalyzes the conversion of shikimate (SKM; Figure 1) into shikimate-3-phosphate (S3P) using adenosine triphosphate (ATP) as co-substrate. The M. tuberculosis shikimate kinase (MtSK) has high sequence similarity, 54%, to the aroK-encoded SK I in Escherichia coli.5 E. coli has a second isozyme, SK II, encoded by aroL, that has a much higher affinity for shikimate than SKI and normally functions in aromatic biosynthesis in the cell.6

Crystal structures of shikimate kinases belonging to the SK I type were determined for a number of organisms including E. coli,7 Campylobacter jejuni,8 Helicobacter pylori9 and M. tuberculosis. The available MtSK structures show the enzyme in a number of liganded states, including binary complexes of MtSK with SO410 or MgADP11 (PDB code 1L4Y), and ternary complexes of MtSK with shikimate (SKM) as one ligand and SO410 (2G1K), ADP12 (1U8A), MgADP,13 or the ATP analogue AMPPCP10 (1ZYU) as a second ligand. Until now, no structural information has been available on the enzyme in unliganded form and in productive liganded states. Putative structures of an “apo-MtSK” and an “MtSK.SKM” complex that were reported recently10 in fact both contained a sulfate ion bound at the nucleotide binding site, which as it is discussed below significantly affects the enzyme conformation.

Here, we describe a number of high-resolution crystal structures of MtSK in a series of different functional states of the enzymatic reaction. These include, in particular, the (unliganded) apo-form of the enzyme, binary complexes of the enzyme with MgATP and with SKM, and a ternary complex with the products shikimate-3-phosphate (S3P) and ADP. Furthermore, we initiated catalytic turnover of shikimate and MgATP by MtSK in the crystalline environment and observed the formation of the products shikimate-3-phosphate and ADP, followed by release of ADP as the first product. On the basis of the crystal structures, which were obtained for the reaction states before initiation of the enzymatic reaction and immediately after the catalytic step, we derived a structural model of the transition state that is consistent with an in-line associative mechanism of phosphoryl transfer. Our approach is complementary to previous studies of phosphoryl transfer mechanisms in phosphokinases that utilized mimics to capture the transition state.14

Section snippets

Overall structure and domain motions

We determined crystal structures of MtSK in the apo state and in a number of different liganded states (Table 1). The structures MtSK·SO4·SKM and MtSK·ADP·SKM are similar to the previously described structures 2G1K and 1U8A, respectively, but have substantially higher resolution. The following discussion is entirely based on the presently reported structures, except for the previously described structures MtSK·MgADP (1L4Y) and MtSK·SKM·AMPPCP (1ZYU) that are referred to by using their PDB codes.

Conclusions

The present crystal structure analysis of MtSK provided snapshots of its interactions with its natural substrates and products that depicted the enzyme conformation in most of the different states adopted during the catalytic cycle. The comparison of the apo-form of the enzyme to binary and ternary complexes with shikimate and nucleotide led to an identification of the associated domain motions. Nucleotide binding induces a change in the P-loop conformation and a rotation of the NB domain

Cloning, expression, and purification

The gene aroK was amplified by PCR using genomic DNA of M. tuberculosis H37Rv and the gene-specific primers aroK5 (forward) TCTCCAAGCTTCATATGGCACCCAAAGCGGTTCTCGT and aroK3 (reverse) GATATCTCGAGTGTGGCCGCCTCGCTGGGGCT. The primers contained HindIII, NdeI (forward) and XhoI (reverse) restriction sites used for cloning of the PCR product into pBluescript KS(-) (Stratagene) and pET-24b(+) (Novagen) vectors. The XhoI site of the reverse primer was placed immediately downstream of the last aroK codon,

Acknowledgements

We thank Birgitta Fried, Susanne Meier and Christian Mönnich for technical assistance in the sample preparation and crystallization, and Dr Galina S. Kachalova for valuable discussions. The work was supported by the Bundesministerium für Bildung und Forschung, BMBF/PTJ, under grant number BIO/0312992A (to H.D.B.).

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