Quaternary Structure Analyses of an Essential Oligomeric Enzyme

Costa, Tatiana P. Soares da; Christensen, Janni; Desbois, Sebastien; Gordon, Shane E.; Gupta, Ruchi; Hogan, Campbell J; Nelson, Tao G; Downton, Matthew T.; Gardhi, Chamodi K.; Abbott, Belinda M.; Wagner, John; Panjikar, Santosh; Perugini, Matthew A.

doi:10.1016/bs.mie.2015.06.020

Cited by 24 publications

(28 citation statements)

References 32 publications

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“…Interestingly, these interactions are also observed for several other enzymes, including dihydrodipicolinate synthase, which catalyzes the first committed and rate-limiting step in the DAP biosynthesis pathway (2,3). For dihydrodipicolinate synthase, dimerization has been shown to be important for completing the active site of each monomer through the interdigitation of a key catalytic triad residue (18,20,22,23,35,40). Similarly, recent studies demonstrate that dimerization is required for the catalytic activity of diaminopimelate epimerase, which also functions in the DAP pathway (16).…”

Section: Discussionmentioning

confidence: 98%

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Peverelli

Costa

Kirby

et al. 2016

Journal of Biological Chemistry

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Diaminopimelate decarboxylase (DAPDC) catalyzes the final step in the diaminopimelate biosynthesis pathway of bacteria. The product of the reaction is the essential amino acid L-lysine, which is an important precursor for the synthesis of the peptidoglycan cell wall, housekeeping proteins, and virulence factors of bacteria. Accordingly, the enzyme is a promising antibacterial target. Previous structural studies demonstrate that DAPDC exists as monomers, dimers, and tetramers in the crystal state. However, the active oligomeric form has not yet been determined. We show using analytical ultracentrifugation, small angle x-ray scattering, and enzyme kinetic analyses in solution that the active form of DAPDC from Bacillus anthracis, Escherichia coli, Mycobacterium tuberculosis, and Vibrio cholerae is a dimer. The importance of dimerization was probed further by generating dimerization interface mutants (N381A and R385A) of V. cholerae DAPDC. Our studies indicate that N381A and R385A are significantly attenuated in catalytic activity, thus confirming that dimerization of DAPDC is essential for function. These findings provide scope for the development of new antibacterial agents that prevent DAPDC dimerization. Diaminopimelate decarboxylase (DAPDC)3 (E.C. 4.1.1.20) is a member of the pyridoxal 5Ј-phosphate (PLP)-dependent decarboxylases (1). It catalyzes the irreversible and stereospecific decarboxylation of meso-diaminopimelate (meso-DAP) in the final step of the diaminopimelate (DAP) biosynthesis pathway of bacteria and plants (2, 3). The product of the reaction, L-lysine, is an important building block for the biosynthesis of the peptidoglycan cell wall, housekeeping proteins, and virulence factors of bacteria. Consequently, DAPDC represents a promising target for the development of novel antibacterial agents (4).A DAPDC reaction mechanism has been previously proposed (5, 6). The reaction mechanism is thought to be initiated by the formation of a Schiff base between PLP and a conserved active site lysine from the (Y/F)AXKA motif (7). The substrate, meso-DAP, then binds and forms a subsequent Schiff base with PLP. This is suggested to be coordinated by the highly conserved CE(S/T)XD motif provided by an adjacent subunit (7). Decarboxylation of meso-DAP is then mediated by the sulfhydryl nucleophile provided by the conserved cysteine of the CE(S/T)XD motif thereby leaving the substrate cradled between two monomers (8). This proposed mechanism suggests DAPDC functions as a dimer.To support this assertion, previous structural studies of DAPDC from Methanocaldococcus jannaschii (PDB codes 1TWI and 1TUF) (9), Aquifex aeolicus (PDB code 2P3E), and Brucella melitensis (PDB code 3VAB) show that the enzyme crystallizes as a homodimer, with each monomer comprised of two domains (9). Domain I forms an ␣/␤ barrel, whereas domain II is comprised of a mixed ␤-sheet flanked by ␣-helices (9). The overall fold is similar to the functionally related enzyme ornithine decarboxylase, which also uses PLP as a cofactor in a decarboxylatio...

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Section: Discussionmentioning

confidence: 98%

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Peverelli

Costa

Kirby

et al. 2016

Journal of Biological Chemistry

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“…Lysine is synthesised de novo in bacteria, plants and some fungi123. This occurs through either the α-aminoadipate pathway in fungi4 or the diaminopimelate pathway in bacteria and plants123.…”

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“…This occurs through either the α-aminoadipate pathway in fungi4 or the diaminopimelate pathway in bacteria and plants123. The diaminopimelate pathway commences with the condensation of pyruvate and ( S )-aspartate semialdehylde (ASA) to form (4 S )-4-hydroxy-2,3,4,5-tetrahydro-(2 S )-dipicolinic acid (HTPA) (Fig.…”

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confidence: 99%

“…The diaminopimelate pathway commences with the condensation of pyruvate and ( S )-aspartate semialdehylde (ASA) to form (4 S )-4-hydroxy-2,3,4,5-tetrahydro-(2 S )-dipicolinic acid (HTPA) (Fig. 1A)12356. This reaction is catalysed by dihydrodipicolinate synthase (DHDPS) (EC4.3.3.7), which is the product of an essential gene in bacteria12789.…”

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“…The canonical bacterial DHDPS structure is a homotetramer best described as a ‘head-to-head’ dimer-of-dimers with four identical (β/α) 8 –barrel subunits (Fig. 1B)3725. The structures of DHDPS from several plant species have also been determined showing that the plant orthologues also form homotetramers but in a ‘back-to-back’ dimer-of-dimers arrangement (Fig.…”

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Structure and Function of Cyanobacterial DHDPS and DHDPR

Christensen

Costa

Faou

et al. 2016

Sci Rep

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Lysine biosynthesis in bacteria and plants commences with a condensation reaction catalysed by dihydrodipicolinate synthase (DHDPS) followed by a reduction reaction catalysed by dihydrodipicolinate reductase (DHDPR). Interestingly, both DHDPS and DHDPR exist as different oligomeric forms in bacteria and plants. DHDPS is primarily a homotetramer in all species, but the architecture of the tetramer differs across kingdoms. DHDPR also exists as a tetramer in bacteria, but has recently been reported to be dimeric in plants. This study aimed to characterise for the first time the structure and function of DHDPS and DHDPR from cyanobacteria, which is an evolutionary important phylum that evolved at the divergence point between bacteria and plants. We cloned, expressed and purified DHDPS and DHDPR from the cyanobacterium Anabaena variabilis. The recombinant enzymes were shown to be folded by circular dichroism spectroscopy, enzymatically active employing the quantitative DHDPS-DHDPR coupled assay, and form tetramers in solution using analytical ultracentrifugation. Crystal structures of DHDPS and DHDPR from A. variabilis were determined at 1.92 Å and 2.83 Å, respectively, and show that both enzymes adopt the canonical bacterial tetrameric architecture. These studies indicate that the quaternary structure of bacterial and plant DHDPS and DHDPR diverged after cyanobacteria evolved.

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Identification of a dimeric KDG aldolase from Agrobacterium tumefaciens

et al. 2017

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Agrobacterium tumefaciens is a Gram-negative bacterium and causative agent of Crown Gall disease that infects a variety of economically important plants. The annotated A. tumefaciens genome contains 10 putative dapA genes, which code for dihydrodipicolinate synthase (DHDPS). However, we have recently demonstrated that only one of these genes (dapA7) encodes a functional DHDPS. The function of the other nine putative dapA genes is yet to be determined. Here, we demonstrate using bioinformatics that the product of the dapA5 gene (DapA5) possesses all the catalytic residues canonical to 2-keto-3-deoxygluconate (KDG) aldolase, which is a class I aldolase involved in glucose metabolism. We therefore expressed, purified, and characterized recombinant DapA5 using mass spectrometry, circular dichroism spectroscopy, analytical ultracentrifugation, and enzyme kinetics. The results show that DapA5 (1) adopts an α/β structure consistent with the TIM-barrel fold of KDG aldolases, (2) possesses KDG aldolase enzyme activity, and (3) exists as a tight dimer in solution. This study shows for the first time that dapA5 from A. tumefaciens encodes a functional dimeric KDG aldolase.

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Quaternary Structure Analyses of an Essential Oligomeric Enzyme

Cited by 24 publications

References 32 publications

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Structure and Function of Cyanobacterial DHDPS and DHDPR

Identification of a dimeric KDG aldolase from Agrobacterium tumefaciens

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