Comparative structural and mechanistic studies of 4-hydroxy-tetrahydrodipicolinate reductases from Mycobacterium tuberculosis and Vibrio vulnificus
Introduction
Studies on the antibiotic-pathogen interactions at the molecular level show that the majority of antibiotics target cell wall synthesis, protein synthesis, and DNA replication and repair [1]. Enzymes containing trans peptidase and trans glycosylase domains, ribosomal machinery and DNA gyrase act as sites of action for antibiotics within these specific pathways [2]. Unfortunately, pathogenic bacteria have developed many defensive mechanisms to evade the antibiotics, including production of β-lactamases, enzymatic modification of drug targets, and development of drug efflux systems [1]. Researchers have identified several approaches to combat rising antibiotic resistance. The most direct involve the development of new classes of antibiotics and compounds that disrupt drug resistance mechanisms [2]. In parallel, several new bacterial components are also being studied as potential novel drug targets. These include bacterial proteases [3], two component signal transduction systems [4], riboswitches [5] and several enzymes involved in fatty acid, nucleotide and amino acid biosynthesis [6]. Disrupting enzymes involved in central biosynthetic pathways will greatly reduce the survival and pathogenicity of bacteria due to the lack of alternative biosynthetic cycles. These will also have an additional advantage of not having homologous enzymes in humans [6]. One such pathway in bacteria is the amino acid lysine or meso-diaminopimelate biosynthetic pathway.
The products of the pathway, meso-diaminopimelate and lysine, are essential for bacterial survival. The meso-diaminopimelate and lysine (for some bacterial species) are essential for bacterial cell shape and rigidity due their role in the covalent linkage of peptidoglycan in the cell wall [7]. Lysine is also an essential amino acid for protein synthesis. Several enzymes from this pathway are currently under study as potential drug targets including aspartate semi aldehyde dehydrogenase (ASADH) [8], 4-hydroxy-tetrahydrodipicolinate synthase (DapA) [9], 4-hydroxy-tetrahydrodipicolinate reductase (DapB) [10], and succinyl diaminopimilate desuccinylase (DapE) [11]. According to the Database of Essential Genes, the genes that code for these enzymes, including DapB, are essential in many pathogenic bacteria (Table S1) [12]. This paper focuses on structural and mechanistic studies of DapB. Functionally, recent work demonstrates that this enzyme catalyzes conversion of (2S, 4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate (HTPA) to 2,3,4,5-tetrahydrodipicolinate in an NADH/NADPH dependent reaction (Fig. 1) and dihydrodipicolinate (DHDP) is not the substrate as previously thought [13].
The structure for DapB has been determined from various bacterial species including Escherichia coli [14], Bartonella henselae [15], Staphylococcus aureus [16], Neisseria gonorrhoeae [10], Thermotoga maritima [17], Coxiella burnetii, and Pseudomonas aeruginosa among others. All of these structures indicate that bacterial DapB is a homotetrameric enzyme. Each monomer can be divided into two functional domains. The first domain is responsible for oligomerization and the second is the NADH/NADPH binding domain (Fig. 2). An interesting feature observed in DapB enzymes that drives the enzyme reaction is the change in conformation upon ligand binding. Binding of pyridine nucleotide or the cofactor induces a change primarily in nucleotide binding domain resulting into partial closure of each monomer (Fig. S1). This change is usually less than 10°. However, binding of both the cofactor and substrate/substrate analogs induces a major conformational change (>30°) resulting into complete closure of the monomer [18]. Even though most DapB homologs can bind both NADH and NADPH, they usually prefer one of the nucleotides. Currently, the reasons for this nucleotide preference remain elusive. In addition, the details of the mechanism for the DapB catalyzed reaction are not known.
This manuscript focuses on DapB homologs from Mycobacterium tuberculosis and Vibrio vulnificus. M. tuberculosis is the causative agent of tuberculosis. It primarily infects the lungs and has been described in a recent report by the World Health Organization as a leading cause of death by a single infectious agent. The incidence of drug resistance reported in this pathogen is extremely high, with over 160 thousand cases documented globally in 2017 [19]. Drug resistant M. tuberculosis has been classified as a serious threat by the Centers for Disease Control and Prevention (USA) [20]. V. vulnificus on the other hand is an opportunistic human pathogen. The natural habitat of this bacterium is coastal or estuarine environments and it is known to colonize shrimp, fish, oysters, and clams [21]. It enters the human body via the consumption of raw seafood. V. vulnificus infection is characterized by gastroenteritis, wound infections, septicemia, and has a very high mortality rate among infected patients [22]. This high mortality rate may be attributed to the drug resistance for many antibiotics including penicillin, ampicillin, and tetracycline seen in the bacterium [23]. Given the high drug resistance observed in both bacteria, research needs to be directed towards identifying new drug targets and inhibitors in order to curb these infections. The manuscript describes the first crystal structures of DapB from V. vulnificus (VvDapB) and several new crystal structures of M. tuberculosis DapB (MtDapB) in apo and ligand bound forms. We have combined the structural analysis and various computational approaches to provide novel insights into mechanism of the DapB catalyzed reaction mechanism.
Section snippets
Screening and identification of potential inhibitors
Differential Scanning Fluorimetry (DSF) assays were performed to identify potential inhibitory compounds for DapB enzymes. The effect of ligands structurally similar to 2,6-pyridine dicarboxylic acid (2,6-PDC), a known inhibitor of DapB enzymes, was evaluated. All the experiments were done using VvDapB as a representative DapB enzyme. Since DapBs share a very high sequence similarity, the results obtained through these experiments may be applicable for other DapB homologs as well [24]. The
Cloning
4-hydroxy-tetrahydrodipicolinate reductase (DapB) genes from Vibrio vulnificus strain CMCP6 (Uniprot: Q8DEM0) (VvDapB) and Mycobacterium tuberculosis HKBS1 (Uniprot: W6GQ56) (MtDapB) were cloned into pJExpress411 plasmid vector by ATUM, Inc. (Newark, CA). Both the genes were codon optimized for expression in E. coli and had an additional N-terminal hexahistidine tag followed by a TEV protease cut site. pJExpress411 plasmid has a selectable kanamycin resistance marker and isopropyl
Author Contributions
MC initiated studies. TB, MC, LD, SK, NJM, SP and LS planned experiments, analyzed data and wrote the manuscript. AKA, LD, TB, SK, VK, NJM and SP performed experiments and/or calculations.
Declaration of Competing Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
X-ray diffraction experimental data was collected at Structural Biology Center (SBC; 19 ID), Life Sciences Collaborative Access Team (LS-CAT; 21 ID) and Southeast Regional Collaborative Access Team (SER-CAT; 22 ID) beamlines at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at www.ser-cat.org/members.html. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under
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