Comparative structural and mechanistic studies of 4-hydroxy-tetrahydrodipicolinate reductases from Mycobacterium tuberculosis and Vibrio vulnificus

https://doi.org/10.1016/j.bbagen.2020.129750Get rights and content

Highlights

  • Lysine biosynthesis pathway is a target for development of new antibiotics.

  • Several crystal structures of DapB were determined.

  • Detailed computational analysis of possible reaction mechanisms was performed.

Abstract

Background

The products of the lysine biosynthesis pathway, meso-diaminopimelate and lysine, are essential for bacterial survival. This paper focuses on the structural and mechanistic characterization of 4-hydroxy-tetrahydrodipicolinate reductase (DapB), which is one of the enzymes from the lysine biosynthesis pathway. DapB catalyzes the conversion of (2S, 4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate (HTPA) to 2,3,4,5-tetrahydrodipicolinate in an NADH/NADPH dependent reaction. Genes coding for DapBs were identified as essential for many pathogenic bacteria, and therefore DapB is an interesting new target for the development of antibiotics.

Methods

We have combined experimental and computational approaches to provide novel insights into mechanism of the DapB catalyzed reaction.

Results

Structures of DapBs originating from Mycobacterium tuberculosis and Vibrio vulnificus in complexes with NAD+, NADP+, as well as with inhibitors, were determined and described. The structures determined by us, as well as currently available structures of DapBs from other bacterial species, were compared and used to elucidate a mechanism of reaction catalyzed by this group of enzymes. Several different computational methods were used to provide a detailed description of a plausible reaction mechanism.

Conclusions

This is the first report presenting the detailed mechanism of reaction catalyzed by DapB.

General significance

Structural data in combination with information on the reaction mechanism provide a background for development of DapB inhibitors, including transition-state analogues.

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

References (60)

  • E. Culp et al.

    Bacterial proteases, untapped antimicrobial drug targets

    J. Antibiot.

    (2016)
  • K.F. Blount et al.

    Riboswitches as antibacterial drug targets

    Nat. Biotech.

    (2006)
  • N.L. Haag et al.

    Potential antibacterial targets in bacterial central metabolism

    Int. J. Adv. Life Sci.

    (2012)
  • A. Wehrmann et al.

    Different modes of diaminopimelate synthesis and their role in cell wall integrity: a study with Corynebacterium glutamicum

    J. Bacteriol.

    (1998)
  • N.J. Mank et al.

    Structure of aspartate β-semialdehyde dehydrogenase from Francisella tularensis

    Acta Crystallogr.

    (2018)
  • A. Garg et al.

    Virtual screening of potential drug-like inhibitors against lysine/DAP pathway of Mycobacterium tuberculosis

    BMC Bioinform.

    (2010)
  • D.M. Gillner et al.

    Lysine biosynthesis in bacteria: a metallodesuccinylase as a potential antimicrobial target

    J. Biol. Inorg. Chem.

    (2013)
  • H. Luo et al.

    DEG 10, an update of the database of essential genes that includes both protein-coding genes and noncoding genomic elements

    Nucleic Acids Res.

    (2014)
  • S.R. Devenish et al.

    NMR studies uncover alternate substrates for dihydrodipicolinate synthase and suggest that dihydrodipicolinate reductase is also a dehydratase

    J. Med. Chem.

    (2010)
  • G. Scapin et al.

    Three-dimensional structure of Escherichia coli dihydrodipicolinate reductase in complex with NADH and the inhibitor 2,6-pyridinedicarboxylate

    Biochemistry

    (1997)
  • A.R. Cala et al.

    The crystal structure of dihydrodipicolinate reductase from the human-pathogenic bacterium Bartonella henselae strain Houston-1 at 2.3Å resolution

    Acta Crystallogr.

    (2016)
  • F.G. Pearce et al.

    Characterization of dihydrodipicolinate reductase from Thermotoga maritima reveals evolution of substrate binding kinetics

    J. Biochem.

    (2008)
  • C.W. Lee et al.

    Crystal structure of dihydrodipicolinate reductase (PaDHDPR) from Paenisporosarcina sp. TG-14: structural basis for NADPH preference as a cofactor

    Sci. Rep.

    (2018)
  • Organization, W. H

    Global Tuberculosis Report 2018

    (2018)
  • Prevention, C. f. D. C. a

    Biggest Threats and Data

    (2018)
  • M.K. Jones et al.

    Vibrio vulnificus: disease and pathogenesis

    Infect Immun..

    (2009)
  • W.T. Booth et al.

    Impact of an N-terminal polyhistidine tag on protein thermal stability

    ACS Omega

    (2018)
  • M. Cirilli et al.

    The three-dimensional structures of the Mycobacterium tuberculosis dihydrodipicolinate reductase-NADH-2,6-PDC and -NADPH-2,6-PDC complexes. Structural and mutagenic analysis of relaxed nucleotide specificity

    Biochemistry

    (2003)
  • R. Janowski et al.

    The structure of dihydrodipicolinate reductase (DapB) from Mycobacterium tuberculosis in three crystal forms

    Acta Crystallogr.

    (2010)
  • G. Qi et al.

    A comprehensive and non-redundant database of protein domain movements

    Bioinformatics

    (2005)
  • Cited by (0)

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