Structure of the Mycobacterium tuberculosis Flavin Dependent Thymidylate Synthase (MtbThyX) at 2.0 Å Resolution

doi:10.1016/j.jmb.2005.07.071

Journal of Molecular Biology

Volume 352, Issue 5, 7 October 2005, Pages 1091-1104

https://doi.org/10.1016/j.jmb.2005.07.071 Get rights and content

A novel flavin-dependent thymidylate synthase was identified recently as an essential gene in many archaebacteria and some pathogenic eubacteria. This enzyme, ThyX, is a potential antibacterial drug target, since humans and most eukaryotes lack the thyX gene and depend upon the conventional thymidylate synthase (TS) for their dTMP requirements. We have cloned and overexpressed the thyX gene (Rv2754c) from Mycobacterium tuberculosis in Escherichia coli. The M. tuberculosis ThyX (MtbThyX) enzyme complements the E. coli χ2913 strain that lacks its conventional TS activity. The crystal structure of the homotetrameric MtbThyX was determined in the presence of the cofactor FAD and the substrate analog, 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdUMP). In the active site, which is formed by three monomers, FAD is bound in an extended conformation with the adenosine ring in a deep pocket and BrdUMP in a closed conformation near the isoalloxazine ring. Structure-based mutational studies have revealed a critical role played by residues Lys165 and Arg168 in ThyX activity, possibly by governing access to the carbon atom to be methylated of a totally buried substrate dUMP.

Introduction

Synthesis of 2′-deoxythymidine-5′-monophosphate (dTMP) is essential in all organisms. Thymidylate synthase (TS; EC 2.1.1.45), the enzyme responsible for this reaction in most eubacteria, plants and eukaryotic cells, has been studied extensively and its catalytic mechanism is well understood.1, 2 Until recently, TS, the translation product of the gene thyA, has been assumed to be responsible for de novo synthesis of dTMP in all organisms. However, recent genomic analyses have revealed that the thyA gene is absent from several bacterial and archaeal genome sequences.³ The search for an alternative source of dTMP has led to the discovery of an alternative thymidylate synthase, thyX, in these genomes.4, 5 The translational product of the thyX gene is referred to as thymidylate synthase complementing protein (TSCP) or as ThyX.

Both the TS and the ThyX enzymes catalyze the conversion of 2′-deoxyuridine-5′-monophosphate (dUMP) to dTMP in the presence of R-N⁵,N¹⁰-methylene-5,6,7,8-tetrahydrofolate (CH₂THF). The TS-catalyzed reaction is a reductive methylation in which CH₂THF acts as both the methylene group donor and the reductant, resulting in 7,8-dihydrofolate (DHF) as the product. TS does not contain a cofactor. In this mechanism, DHF production is coupled to dihydrofolate reductase (DHFR; EC 1.5.1.3) to generate tetrahydrofolate (THF), which in turn is methylated by serinehydroxymethyl transferase (SHMT; EC 2.1.2.1) to regenerate CH₂THF (Scheme 1). However, in the ThyX-catalyzed reaction, CH₂THF is used only as a methylene group donor and, therefore, the product is THF. Accordingly, organisms that lack the gene for TS also lack the gene for DHFR.³ In ThyX catalysis, FAD fulfils the role as reductant and is bound to the enzyme as the cofactor (Scheme 1). Electron transfer from reduced pyridine nucleotides (NADH or NADPH) to flavin is the critical first step in ThyX catalysis.4, 6 Thus, ThyX provides an alternative mechanism for the production of dTMP.

ThyX is intriguing in biochemical and mechanistic aspects, but its potential as an antibacterial drug target is of exceptional importance as well. The thyX gene is rare in eukaryotes and absent from humans. Several human pathogens, including Helicobacter pylori, depend exclusively on the ThyX enzyme for their dTMP requirements, suggesting that inhibitors of ThyX activity will be potent drugs against these pathogens.4, 6 While the importance of ThyX in these organisms is obvious, several Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium leprae, present an interesting and challenging situation. The Mycobacteria possess highly conserved genes for both the classical TS and the ThyX enzymes. Thus, it is not clear whether either gene is essential. However, thyX was identified in a screen for essential genes in M. tuberculosis.7, 8 One interesting explanation for the duality is that the Mycobacteria might preferentially use either TS or ThyX under different growth conditions, as has been postulated for the two glucose-6-phosophate dehydrogenase enzymes.⁹

As a part of our long-standing efforts to study potential drug targets in global human pathogens, we have expressed the gene Rv2754c from M. tuberculosis and functionally characterized its protein product, ThyX (MtbThyX). Furthermore, we have determined the structure of an Ile65Met/Leu175Met double mutant of MtbThyX at 2.0 Å resolution in the presence of the cofactor FAD and the substrate analog 5-bromo-2′-deoxyuridine-5′-monophosphate (BrdUMP). This represents the first structure of a ThyX from a human pathogen. The role of several residues in catalysis was confirmed by mutation and testing whether the mutant ThyX complemented the TS defect in the χ2913 strain of Escherichia coli. The structure provides new insights into the mechanism of ThyX catalysis and reveals the architecture of the active site that might be exploited in the design of pathogen-specific inhibitors.

Section snippets

Cloning of MtbThyX

The gene that encodes the M. tuberculosis ThyX protein was identified by its homology to the H. pylori and T. maritima proteins.4, 10 The enzymatic activity of both the wild-type and the I65M/L175M double mutant was confirmed by testing whether the MtbThyX protein could complement the defect in thymidylate synthesis of the E. coli strain, in TS-deficient χ2913 E. coli in which the thyA gene has been deleted (Figure 1). Although wild-type E. coli depends exclusively on its conventional TS, thyA, the

MtbThyX structure and catalytic mechanism

While the catalytic mechanism of the classical TS (ThyA) is understood in great detail,1, 2 much less is known about the mechanism of the ThyX-catalyzed reaction. The biochemical studies on the ThyX enzyme from various sources have suggested different mechanisms. The ThyX enzymes from T. maritima and Chlamydia trachomatis (CtThyX) have been suggested to follow a ping-pong mechanism.12, 14 However, a sequential mechanism is suggested for the Paramecium bursaria Chlorella virus-1 enzyme.¹⁵

In the

Conclusions

We describe here the first structure of an FAD-dependent TS enzyme determined from a eubacterial source, M. tuberculosis, which is the major global bacterial infectious agent at present. The structural, functional and genomic comparisons that we have documented support the idea that the ThyX enzymes are strongly conserved, even among evolutionarily distant organisms. Since several of these distantly related human pathogens depend exclusively on ThyX activity for their dTMP requirements, design

Cloning M. tuberculosis ThyX into the pUC vector

To isolate the thyX gene from M. tuberculosis, we performed a BLASTp search on the H. pylori ThyX protein sequence against the complete genome of M. tuberculosis H37Rv. The predicted gene, Rv2754c, was identified by its homology to H. pylori ThyX.⁴ Primers were designed to bind specifically to the 5′ and 3′ ends of the Rv2754c gene (Table 5). The forward primer (pUC18 forward) contains an EcoRI site, and the reverse primer (pUC18 reverse) a SalI site, both of which are just 5′ of the respective

Acknowledgements

The authors gratefully acknowledge the support provided by the staff at beam lines 8.2.1 and 8.2.2 at ALS, and at SBC-CAT 19-ID beam line at APS. We thank Brian Krumm, Jan Abendroth, Jungpeng Deng, Robert Lucas, Konstantin Korotkov, Mark Robien and Juergen Bosch at various stages of the purification, data collection and structure determination. We thank Francis Athapilly for maintaining the computer hardware and software environment. A grant from the Puget Sound Partners for Global Health to

References (32)

H. Myllykallio et al.
Life without dihydrofolate reductase FolA
Trends Microbiol.
(2003)
M. Giladi et al.
Genetic evidence for a novel thymidylate synthase in the halophilic archaeon Halobacterium salinarum and in Campylobacter jejuni
FEMS Microbiol. Letters
(2002)
I.I. Mathews et al.
Functional analysis of substrate and cofactor complex structures of a thymidylate synthase-complementing protein
Structure (Camb)
(2003)
E. Purwantini et al.
Presence of F420-dependent glucose-6-phosphate dehydrogenase in Mycobacterium and Nocardia species, but absence from Streptomyces and Corynebacterium species and methanogenic Archaea
FEMS Microbiol. Letters
(1997)
J. Griffin et al.
Catalytic mechanism of Chlamydia trachomatis flavin-dependent thymidylate synthase
J. Biol. Chem.
(2005)
S. Graziani et al.
Functional analysis of FAD-dependent thymidylate synthase ThyX from Paramecium bursaria Chlorella virus-1
J. Biol. Chem.
(2004)
G.D. Van Duyne et al.
Atomic structures of the human immunophilin FKBP-12 complexes with FK506 and rapamycin
J. Mol. Biol.
(1993)
Z. Otwinowski et al.
Processing of X-ray diffraction data collected in oscillation mode
Methods Enzymol.
(1997)
C.W. Carreras et al.
The catalytic mechanism and structure of thymidylate synthase
Annu. Rev. Biochem.
(1995)
J.S. Finer-Moore et al.
Lessons and conclusions from dissecting the mechanism of a bisubstrate enzyme: thymidylate synthase mutagenesis, function, and structure
Biochemistry
(2003)

H. Myllykallio et al.

An alternative flavin-dependent mechanism for thymidylate synthesis

Science

(2002)

J. Rengarajan et al.

From The Cover: genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages

Proc. Natl Acad. Sci. USA

(2005)

C.M. Sassetti et al.

Genes required for mycobacterial growth defined by high density mutagenesis

Mol. Microbiol.

(2003)

P. Kuhn et al.

Crystal structure of thy1, a thymidylate synthase complementing protein from Thermotoga maritima at 2.25 Å resolution

Proteins: Struct. Funct. Genet.

(2002)

D. Leduc et al.

Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers

Proc. Natl Acad. Sci. USA

(2004)

N. Agrawal et al.

Mechanistic studies of a flavin-dependent thymidylate synthase

Biochemistry

(2004)

Cited by (64)

Mechanism of Naphthoquinone Selectivity of Thymidylate Synthase ThyX
2020, Biophysical Journal
Naphthoquinones (NQs) are natural and synthetic compounds with a wide range of biological activities commonly attributed to their redox activity and/or chemical reactivity. However, genetic and biochemical experiments have recently demonstrated that 2-hydroxy-NQs (2-OH-NQs) act as highly specific noncovalent inhibitors of the essential bacterial thymidylate synthase ThyX in a cellular context. We used biochemical experiments and molecular dynamics simulations to elucidate the selective inhibition mechanism of NQ inhibitors of ThyX from Mycobacterium tuberculosis (Mtb). Free energy simulations rationalized how ThyX recognizes the natural substrate dUMP in the N3-ionized form using an arginine, Arg199, in Mtb. The results further demonstrated that 2-OH-NQ, similar to dUMP, binds to ThyX in the ionized form, and the strong and selective binding of 2-OH-NQ to ThyX is also explained by electrostatic interactions with Arg199. The stronger binding of the close analog 5F-dUMP to ThyX and its inhibitory properties compared with dUMP were explained by the stronger acidity of the uracil N3 atom. Our results, therefore, revealed that the ionization of 2-OH-NQs drives their biological activities by mimicking the interactions with the natural substrate. Our observations encourage the rational design of optimized ThyX inhibitors that ultimately may serve as antibiotics.
Molecular modeling of drug-pathophysiological Mtb protein targets: Synthesis of some 2-thioxo-1, 3-thiazolidin-4-one derivatives as anti-tubercular agents
2017, Journal of Molecular Structure
Twenty novel 2-thioxo-1, 3-thiazolidin-4-one derivatives (5a-5t) were synthesized and evaluated for their antitubercular activity. The structure of the compounds was confirmed by IR, NMR and Mass Spectroscopy methods. In addition, single-crystal X-ray diffraction was performed for compound 5a. All the synthesized compounds were screened for their in-vitro antimycobacterial activity against MTB (H37RV, ATCC No: 27294) by Alamar Blue assay method. Compounds 5r, 5k, 5t displayed most potent in-vitro activity with MICs of 0.05, 0.1, 0.2 μg/ml concentrations respectively which are comparatively potent than the standards. Molecular docking and dynamics simulations were performed to find out the plausible mechanism of the titled compounds.
Flavin-dependent thymidylate synthase: N5 of flavin as a Methylene carrier
2017, Archives of Biochemistry and Biophysics
Thymidylate is synthesized de novo in all living organisms for replication of genomes. The chemical transformation is reductive methylation of deoxyuridylate at C5 to form deoxythymidylate. All eukaryotes including humans complete this well-understood transformation with thymidylate synthase utilizing 6R-N⁵-N¹⁰-methylene-5,6,7,8-tetrahydrofolate as both a source of methylene and a reducing hydride. In 2002, flavin-dependent thymidylate synthase was discovered as a new pathway for de novo thymidylate synthesis. The flavin-dependent catalytic mechanism is different than thymidylate synthase because it requires flavin as a reducing agent and methylene transporter. This catalytic mechanism is not well-understood, but since it is known to be very different from thymidylate synthase, there is potential for mechanism-based inhibitors that can selectively inhibit the flavin-dependent enzyme to target many human pathogens with low host toxicity.
Discovery of a new Mycobacterium tuberculosis thymidylate synthase X inhibitor with a unique inhibition profile
2017, Biochemical Pharmacology
Tuberculosis (TB), mainly caused by Mycobacterium tuberculosis (Mtb), is an infection that is responsible for roughly 1.5 million deaths per year. The situation is further complicated by the wide-spread resistance to the existing first- and second-line drugs. As a result of this, it is urgent to develop new drugs to combat the resistant bacteria as well as have lower side effects, which can promote adherence to the treatment regimens. Targeting the de novo synthesis of thymidylate (dTMP) is an important pathway to develop drugs for TB. Although Mtb carries genes for two families of thymidylate synthases (TS), ThyA and ThyX, only ThyX is essential for its normal growth. Both enzymes catalyze the conversion of uridylate (dUMP) to dTMP but employ a different catalytic approach and have different structures. Also, ThyA is the only TS found in humans. This is the rationale for identifying selective inhibitors against ThyX. We exploited the NADPH oxidation to NADP⁺ step, catalyzed by ThyX, to develop a spectrophotometric biochemical assay. Success of the assay was demonstrated by its effectiveness (average Z′ = 0.77) and identification of selective ThyX inhibitors. The most potent compound is a tight-binding inhibitor with an IC₅₀ of 710 nM. Its mechanism of inhibition is analyzed in relation to the latest findings of ThyX mechanism and substrate and cofactor binding order.
Perturbation of genome integrity to fight pathogenic microorganisms
2017, Biochimica et Biophysica Acta - General Subjects
Resistance against antibiotics is unfortunately still a major biomedical challenge for a wide range of pathogens responsible for potentially fatal diseases.
In this study, we aim at providing a critical assessment of the recent advances in design and use of drugs targeting genome integrity by perturbation of thymidylate biosynthesis.
We find that research efforts from several independent laboratories resulted in chemically highly distinct classes of inhibitors of key enzymes within the routes of thymidylate biosynthesis. The present article covers numerous studies describing perturbation of this metabolic pathway in some of the most challenging pathogens like Mycobacterium tuberculosis, Plasmodium falciparum, and Staphylococcus aureus.
Our comparative analysis allows a thorough summary of the current approaches to target thymidylate biosynthesis enzymes and also include an outlook suggesting novel ways of inhibitory strategies. This article is part of a Special Issue entitled “Science for Life” Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo
Thermal stability and binding energetics of thymidylate synthase ThyX
2016, International Journal of Biological Macromolecules
Citation Excerpt :
The wild type (WT) ThyX proteins from Thermotoga maritima (TmThyX) and Paramecium bursaria chlorella virus-1 WT (PBCV-1 ThyX), studied in this work, show 53% amino acid identity [4]. The amino acid residues involved in the binding of FAD and dUMP to ThyX have been identified in the crystallographic structure of the ternary complexes [5,6]. The conformation of key active site residues of PBCV-1 ThyX differs from that of TmThyX structures, suggesting different structural changes during catalysis [7].
The bacterial thymidylate synthase ThyX is a multisubstrate flavoenzyme that takes part in the de novo synthesis of thymidylate in a variety of microorganisms. Herein we study the effect of FAD and dUMP binding on the thermal stability of wild type (WT) ThyX from the mesophilic Paramecium bursaria chlorella virus-1 (PBCV-1) and from the thermophilic bacterium Thermotoga maritima (TmThyX), and from two variants of TmThyX, Y91F and S88W, using differential scanning calorimetry. The energetics underlying these processes was characterized by isothermal titration calorimetry. The PBCV-1 protein is significantly less stable against the thermal challenge than the TmThyX WT. FAD exerted stabilizing effect greater for PBCV-1 than for TmThyX and for both mutants, whereas binding of dUMP to FAD-loaded proteins stabilized further only TmThyX. Different thermodynamic signatures describe the FAD binding to the WT ThyX proteins. While TmThyX binds FAD with a low μM binding affinity in a process characterized by a favorable entropy change, the assembly of PBCV-1 with FAD is governed by a large enthalpy change opposed by an unfavorable entropy change resulting in a relatively strong nM binding. An enthalpy-driven formation of a high affinity ternary ThyX/FAD/dUMP complex was observed only for TmThyX.

View all citing articles on Scopus

View full text

Journal of Molecular Biology

Structure of the Mycobacterium tuberculosis Flavin Dependent Thymidylate Synthase (MtbThyX) at 2.0 Å Resolution

Introduction

Section snippets

Cloning of MtbThyX

MtbThyX structure and catalytic mechanism

Conclusions

Cloning M. tuberculosis ThyX into the pUC vector

Acknowledgements

Trends Microbiol.

FEMS Microbiol. Letters

Structure (Camb)

FEMS Microbiol. Letters

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

Methods Enzymol.

The catalytic mechanism and structure of thymidylate synthase

Annu. Rev. Biochem.

Lessons and conclusions from dissecting the mechanism of a bisubstrate enzyme: thymidylate synthase mutagenesis, function, and structure

Biochemistry

An alternative flavin-dependent mechanism for thymidylate synthesis

Science

From The Cover: genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages

Proc. Natl Acad. Sci. USA

Genes required for mycobacterial growth defined by high density mutagenesis

Mol. Microbiol.

Crystal structure of thy1, a thymidylate synthase complementing protein from Thermotoga maritima at 2.25 Å resolution

Proteins: Struct. Funct. Genet.

Functional evidence for active site location of tetrameric thymidylate synthase X at the interphase of three monomers

Proc. Natl Acad. Sci. USA

Mechanistic studies of a flavin-dependent thymidylate synthase

Biochemistry