Biochemical studies and crystal structure determination of dihydrodipicolinate synthase from Pseudomonas aeruginosa

Kaur, Navpreet; Gautam, Ankur; Kumar, Sanjit; Singh, Avinash; Singh, Nagendra; Sharma, Sujata; Sharma, Rohit K.; Tewari, Rupinder

doi:10.1016/j.ijbiomac.2011.03.002

Cited by 29 publications

(37 citation statements)

References 36 publications

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“…The crystal structures of DHDPS from several species have been solved including enzymes from the thermophilic methanogenic archaea, Methanocaldococcus jannaschii (Mj) (Padbanabhan et al 2009), the gram-negative bacteria like Thermotoga maritima (Tm) (Pearce et al 2011), Campylobacter jejuni (Cj, Yet to be published), E. coli (Ec) (Mirwaldt et al 1995), Pseudomonas aeruginosa (Ps) (Kaur et al 2011), Mycobacterium tuberculosis (Mt) (Kefala et al 2008), and Gram-positive bacteria like Bacillus anthracis (Ba) (Voss et al 2010) and Staphylococcus aureus (Sa) (Girish et al 2008). From the plant sources, the DHDPS structure is available for Nicotiana sylvestris (Ns) (Blickling et al 1997), Pivus sativum (Psa) (Dereppe et al 1992), Arabidopsis thaliana (At) , Vitis vinifera (Vv), and Agrobacterium tumefaciens (Atu) (Atkinson et al 2012a, b).…”

Section: Introductionmentioning

confidence: 99%

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus

et al. 2014

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Dihydrodipicolinate synthase (DHDPS, E.C.4.2.1.52) catalyzes the first committed step in the lysine biosynthetic pathway: the condensation of (S)-aspartate semialdehyde and pyruvate to form (4S)-4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid. Since (S)-lysine biosynthesis does not occur in animals, DHDPS is an attractive target for rational antibiotic and herbicide design. Here, we report the crystal structure of DHDPS from a hyperthermophilic bacterium Aquifex aeolicus (AqDHDPS). L-Lysine is used as an important animal feed additive where the production is at the level of 1.5 million tons per year. The biotechnological manufacture of lysine has been going for more than 50 years which includes over synthesis and reverse engineering of DHDPS. AqDHDPS revealed a unique disulfide linkage which is not conserved in the homologues of AqDHDPS. In silico mutation of C139A and intermolecular ion-pair residues and the subsequent molecular dynamics simulation of the mutants showed that these residues are critical for the stability of AqDHDPS tetramer. MD simulations of AqDHDPS at three different temperatures (303, 363 and 393 K) revealed that the molecule is stable at 363 K. Thus, this structural and in silico study of AqDHDPS likely provides additional details towards the rational and structure-based design of hyper-L-lysine producing bacterial strains.

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Section: Introductionmentioning

confidence: 99%

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus

et al. 2014

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“…This rate-limiting step is catalysed by the enzyme DHDPS. In all Gram-negative pathogens (Dobson, Devenish et al, 2005;Phenix & Palmer, 2008;Devenish et al, 2009;Kaur et al, 2011) and plants (Matthews & Widholm, 1979;Wallsgrove & Mazelis, 1980;Kumpaisal et al, 1987;Dereppe et al, 1992;Blickling, Beisel et al, 1997;Griffin et al, 2012;Atkinson et al, 2013) studied to date, lysine acts as a feedback inhibitor to DHDPS.…”

Section: Introductionmentioning

confidence: 99%

“…The structure of DHDPS has been investigated in several bacteria including Agrobacterium tumefaciens , Bacillus anthracis (Blagova et al, 2006;Voss et al, 2009Voss et al, , 2010, Clostridium botulinum , Corynebacterium glutamicum (Rice et al, 2008), Escherichia coli (Mirwaldt et al, 1995;Dobson, Griffin et al, 2005), Hahella chejuensis (Kang et al, 2010), Methanococcus jannaschii (Padmanabhan et al, 2009), Mycobacterium tuberculosis (Kefala et al, 2008;Evans et al, 2011), Neisseria meningitidis (Devenish et al, 2009), Pseudomonas aeruginosa (Kaur et al, 2011), Shewenella benthica (Wubben et al, 2010), Staphylococcus aureus (Burgess et al, 2008;Girish et al, 2008), Streptococcus pneumoniae (Sibarani et al, 2010) and Thermotoga maritima (Pearce et al, 2006). The enzyme typically forms a homotetramer that is described as a dimer of dimers.…”

Section: Introductionmentioning

confidence: 99%

“…Two such dimers then unite to create a tetramer via interactions at the 'weak-dimer' interface where weaker bonding interactions exist (Perugini et al, 2005;Griffin et al, 2008Griffin et al, , 2010. Recently, homodimers and dimeric mutants of DHDPS have also surfaced that exhibit high levels of activity (Burgess et al, 2008;Girish et al, 2008;Wubben et al, 2010;Evans et al, 2011;Kaur et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

“…This gives rise to four active sites in a tetrameric DHDPS or two active sites in a dimeric DHDPS (Mirwaldt et al, 1995). Adjacent to the active sites is a cleft at the tight-dimer interface to which lysine binds in order to inhibit the enzyme (Dobson, Griffin et al, 2005;Kaur et al, 2011;Griffin et al, 2012;Atkinson et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Cloning to crystallization of dihydrodipicolinate synthase from the intracellular pathogenLegionella pneumophila

et al. 2013

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Dihydrodipicolinate synthase (DHDPS) catalyses the rate-limiting step in the biosynthesis of meso-diaminopimelate and lysine. Here, the cloning, expression, purification and crystallization of DHDPS from the intracellular pathogen Legionella pneumophila are described. Crystals grown in the presence of highmolecular-weight PEG precipitant and magnesium chloride were found to diffract beyond 1.65 Å resolution. The crystal lattice belonged to the hexagonal space group P6 1 22, with unit-cell parameters a = b = 89.31, c = 290.18 Å , and contained two molecules in the asymmetric unit. The crystal structure was determined by molecular replacement using a single chain of Pseudomonas aeruginosa DHDPS as the search model.

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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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Biochemical studies and crystal structure determination of dihydrodipicolinate synthase from Pseudomonas aeruginosa

Cited by 29 publications

References 36 publications

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus

Crystal structure and in silico studies of dihydrodipicolinate synthase (DHDPS) from Aquifex aeolicus

Cloning to crystallization of dihydrodipicolinate synthase from the intracellular pathogenLegionella pneumophila

Identification of thebona fideDHDPS from a common plant pathogen

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