Crystal structure of dihydrodipicolinate synthase from Hahella chejuensis at 1.5 Å resolution

Kang, Beom Sik; Kim, Yeon-Gil; Ahn, Joo‐Myung; Kim, Kyungjin

doi:10.1016/j.ijbiomac.2010.03.005

Cited by 8 publications

(5 citation statements)

References 23 publications

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“…To provide insight into the mechanism of pyruvate-mediated oligomer stabilization, we determined the X-ray crystal structures of Cbot-DHDPS in its native form (2.34 Å resolution, PDB: 3IRD) and in complex with pyruvate (1.19 Å resolution, PDB: 3A5F) (Table 1). Consistent with observations from the aforementioned solution studies, Cbot-DHDPS was found to be a tetramer in the crystalline state (Figure 5A), and of similar architecture to DHDPS enzymes from other bacteria (Atkinson et al, 2014;Blagova et al, 2006;Blickling et al, 1997b;Christensen et al, 2016;Devenish et al, 2009;Dobson et al, 2005b;Dogovski et al, 2013;Kang et al, 2010;Kefala et al, 2008;Mirwaldt et al, 1995;Padmanabhan et al, 2009;Pearce et al, 2006;Perugini et al, 2005;Phenix et al, 2008;Rice et al, 2008;Skovpen et al, 2016;Soares da Costa et al, 2016;Sowole et al, 2016;Sridharan et al, 2014;Voss et al, 2010;Wolterink-van Loo et al, 2008). The monomeric unit of the enzyme comprises a (b/a) 8 barrel with a three helical C-terminal bundle consistent with other DHDPS structures determined to date (Figure 5B).…”

Section: Comparison Of Native and Pyruvate-bound Crystal Structures Of Cbot-dhdpssupporting

confidence: 87%

“…Previous studies suggest that the quaternary structure of DHDPS evolved to optimize protein dynamics for function (Burgess et al, 2008;Griffin et al, 2008Griffin et al, , 2010Muscroft-Taylor et al, 2010;Pearce et al, 2008Pearce et al, , 2011Reboul et al, 2012;Soares da Costa et al, 2015). Although dimeric forms of the enzyme have been described (Burgess et al, 2008;Girish et al, 2008;Kaur et al, 2011), structural studies demonstrate that DHDPS from plants (Atkinson et al, 2012(Atkinson et al, , 2013Blickling et al, 1997a;Griffin et al, 2012) and most bacteria (Atkinson et al, 2014;Blagova et al, 2006;Christensen et al, 2016;Devenish et al, 2009;Dobson et al, 2005b;Dogovski et al, 2009Dogovski et al, , 2012Kang et al, 2010;Kefala et al, 2008;Mirwaldt et al, 1995;Padmanabhan et al, 2009;Pearce et al, 2006;Perugini et al, 2005;Phenix et al, 2008;Rice et al, 2008;Skovpen et al, 2016;Soares da Costa et al, 2015Sowole et al, 2016;Sridharan et al, 2014;Voss et al, 2010;Wolterink-van Loo et al, 2008) assemble as a homotetramer (Figures 1B and 1C). The tetramer is important for catalytic activity, since dimeric forms of bacterial DHDPS engineered by mutating the tetramerizat...…”

Section: Introductionmentioning

confidence: 99%

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Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target

et al. 2018

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Protein dynamics manifested through structural flexibility play a central role in the function of biological molecules. Here we explore the substrate-mediated change in protein flexibility of an antibiotic target enzyme, Clostridium botulinum dihydrodipicolinate synthase. We demonstrate that the substrate, pyruvate, stabilizes the more active dimer-of-dimers or tetrameric form. Surprisingly, there is little difference between the crystal structures of apo and substrate-bound enzyme, suggesting protein dynamics may be important. Neutron and small-angle X-ray scattering experiments were used to probe substrate-induced dynamics on the sub-second timescale, but no significant changes were observed. We therefore developed a simple technique, coined protein dynamics-mass spectrometry (ProD-MS), which enables measurement of time-dependent alkylation of cysteine residues. ProD-MS together with X-ray crystallography and analytical ultracentrifugation analyses indicates that pyruvate locks the conformation of the dimer that promotes docking to the more active tetrameric form, offering insight into ligand-mediated stabilization of multimeric enzymes.

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Section: Comparison Of Native and Pyruvate-bound Crystal Structures Of Cbot-dhdpssupporting

confidence: 87%

Section: Introductionmentioning

confidence: 99%

Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target

et al. 2018

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“…The origin of the enhanced thermal stability is clearly revealed in the 1.9 Å resolution crystal structure of Sp -DHDPS (Figure 7) that shows a tetrameric structure of the enzyme, consistent with our solution studies (Table 4, Figures 4 and 5). The tertiary and quaternary structure architecture of Sp -DHDPS is very similar to Ec -DHDPS [25,31,34] with an overall RMSD for superposition of the tetramers of 1.1 Å (alpha carbon atoms), as well as to other structurally characterized bacterial DHDPS enzymes [16-19,22,24-27,29-36];. However, significant structural differences are observed between Sp -DHDPS and Ec -DHDPS at the subunit interfaces (Figure 9).…”

Section: Discussionmentioning

confidence: 99%

From Knock-Out Phenotype to Three-Dimensional Structure of a Promising Antibiotic Target from Streptococcus pneumoniae

et al. 2013

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Given the rise in drug-resistant Streptococcus pneumoniae, there is an urgent need to discover new antimicrobials targeting this pathogen and an equally urgent need to characterize new drug targets. A promising antibiotic target is dihydrodipicolinate synthase (DHDPS), which catalyzes the rate-limiting step in lysine biosynthesis. In this study, we firstly show by gene knock out studies that S. pneumoniae (sp) lacking the DHDPS gene is unable to grow unless supplemented with lysine-rich media. We subsequently set out to characterize the structure, function and stability of the enzyme drug target. Our studies show that sp-DHDPS is folded and active with a k cat = 22 s-1, K M PYR = 2.55 ± 0.05 mM and K M ASA = 0.044 ± 0.003 mM. Thermal denaturation experiments demonstrate sp-DHDPS exhibits an apparent melting temperature (T M app) of 72 °C, which is significantly greater than Escherichia coli DHDPS (Ec-DHDPS) (T M app = 59 °C). Sedimentation studies show that sp-DHDPS exists in a dimer-tetramer equilibrium with a K D 4→2 = 1.7 nM, which is considerably tighter than its E. coli ortholog (K D 4→2 = 76 nM). To further characterize the structure of the enzyme and probe its enhanced stability, we solved the high resolution (1.9 Å) crystal structure of sp-DHDPS (PDB ID 3VFL). The enzyme is tetrameric in the crystal state, consistent with biophysical measurements in solution. Although the sp-DHDPS and Ec-DHDPS active sites are almost identical, the tetramerization interface of the s. pneumoniae enzyme is significantly different in composition and has greater buried surface area (800 Å2) compared to its E. coli counterpart (500 Å2). This larger interface area is consistent with our solution studies demonstrating that sp-DHDPS is considerably more thermally and thermodynamically stable than Ec-DHDPS. Our study describe for the first time the knock-out phenotype, solution properties, stability and crystal structure of DHDPS from S. pneumoniae, a promising antimicrobial target.

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“…Interestingly, the genome of A. tumefaciens has been sequenced and reveals 10 putative dapA genes that potentially encode DHDPS. Besides rare reports of two dapA genes in Bacillus anthracis and Lactobacillus plantarum and three genes in Hahella chejuensis and Halobacillus halophilus , most bacterial species have a single dapA gene. Therefore, Agrobacterium tumefaciens is the only bacterial species whose genome has been sequenced to date to harbor more than three putative dapA genes.…”

Section: Introductionmentioning

confidence: 99%

Identification of thebona fideDHDPS from a common plant pathogen

et al. 2014

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Agrobacterium tumefaciens is a Gram-negative soil-borne bacterium that causes Crown Gall disease in many economically important crops. The absence of a suitable chemical treatment means there is a need to discover new anti-Crown Gall agents and also characterize bona fide drug targets. One such target is dihydrodipicolinate synthase (DHDPS), a homo-tetrameric enzyme that catalyzes the committed step in the metabolic pathway yielding meso-diaminopimelate and lysine. Interestingly, there are 10 putative DHDPS genes annotated in the A. tumefaciens genome, including three whose structures have recently been determined (PDB IDs: 3B4U, 2HMC, and 2R8W). However, we show using quantitative enzyme kinetic assays that nine of the 10 dapA gene products, including 3B4U, 2HMC, and 2R8W, lack DHDPS function in vitro. A sequence alignment showed that the product of the dapA7 gene contains all of the conserved residues known to be important for DHDPS catalysis and allostery. This gene was cloned and the recombinant product expressed and purified. Our studies show that the purified enzyme (i) possesses DHDPS enzyme activity, (ii) is allosterically inhibited by lysine, and (iii) adopts the canonical homo-tetrameric structure in both solution and the crystal state. This study describes for the first time the structure, function and allostery of the bona fide DHDPS from A. tumefaciens, which offers insight into the rational design of pesticide agents for combating Crown Gall disease.

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Crystal structure of dihydrodipicolinate synthase from Hahella chejuensis at 1.5 Å resolution

Cited by 8 publications

References 23 publications

Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target

Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target

From Knock-Out Phenotype to Three-Dimensional Structure of a Promising Antibiotic Target from Streptococcus pneumoniae

Identification of thebona fideDHDPS from a common plant pathogen

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