Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis

Hor, Lilian; Dobson, Renwick C. J.; Downton, Matthew T.; Wagner, John; Hutton, Craig A.; Perugini, Matthew A.

doi:10.1074/jbc.m113.450148

Cited by 43 publications

(43 citation statements)

References 53 publications

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“…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). Additionally, dimerization has been proposed to be required for reorientating the active site in the activation of apoptotic initiator caspase 9 (41).…”

Section: Discussionmentioning

confidence: 99%

“…Mass Spectrometry-To determine the molar mass of purified recombinant DAPDC enzymes, electrospray ionization time-of-flight mass spectrometry was performed as previously described by Hor et al (16). Recombinant proteins were also subjected to trypsin digestion and the resulting tryptic fragments were sequenced by tandem mass spectrometry as reported previously (17).…”

Section: Methodsmentioning

confidence: 99%

“…When sedimentation equilibrium was attained (ϳ24 h at each speed), data were collected from 6.7 to 7.3 cm using a 0.001-cm step size with 10 averages. The resulting dual speed sedimentation equilibrium data at multiple protein concentrations were fitted globally to various self-association models, including a monomer-dimer model (16,23), using SEDPHAT software (27).…”

Section: Methodsmentioning

confidence: 99%

“…Resulting data were corrected for photomultiplier voltage differences and normalized to pseudo-absorbance values. The pseudo-absorbance profiles were then fitted to various self-association models, including a monomer-dimer model (16,23), using SEDPHAT software (27).…”

Section: Methodsmentioning

confidence: 99%

“…7C). A distinct upward curvature is observed between 5 and 500 nM, which is indicative of a self-associating enzyme where the lower order species is significantly less active than the higher order oligomer (16,20,23). By contrast, the wild-type enzyme displays a linear rate versus concentration relationship at concentrations (5-50 nM) well above the K D 231 (Fig.…”

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confidence: 95%

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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: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 95%

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Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Peverelli

Costa

Kirby

et al. 2016

Journal of Biological Chemistry

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Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

Bearne

2020

Chemistry A European J

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Unlike most enzymes, which exhibit stereospecific substrate binding, racemases and epimerases bind and catalyze the reversible interconversion of enantiomeric and epimeric pairs of substrates. Over the past 15 years, a growing number of racemase and epimerase structures have been solved, furnishing insights into the nature of chiral recognition of substrates by these enzymes. Those enzymes catalyzing stereoinversion of a carbon acid substrate through a direct 1,1‐proton transfer mechanism all bind their substrates in a mirror‐image packing orientation. This does not apply generally to racemases and epimerases that use other mechanisms, such as NADH‐dependent epimerases that employ a “flipping” mechanism. In general, polar groups are bound and fixed at the three binding determinants on the protein defining a pseudo‐mirror plane, while nonpolar groups may be mobile. The hydrogen atoms on each stereocenter are positioned antipodal with respect to the pseudo‐mirror plane, making a two‐base mechanism imperative. Recognition that mirror‐image packing is the common binding mode for enantiomeric or epimeric substrates of these enzymes should inform modelling/docking studies and protein engineering.

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In silico prediction, molecular docking and binding studies of acetaminophen and dexamethasone to Enterococcus faecalis diaminopimelate epimerase

Singh

Das

Yadav

et al. 2021

J of Molecular Recognition

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Enterococcus faecalis (E. faecalis) is a Gram‐positive coccoid, non‐sporulating, facultative anaerobic, multidrug resistance bacterium responsible for almost 65% to 80% of all enterococcal nosocomial infections. It usually causes infective endocarditis, urinary tract and surgical wound infections. The increase in E. faecalis resistance to conventionally available antibiotic has rekindled intense interest in developing useful antibacterial drugs. In E. faecalis, diaminopimelate epimerase (DapF) is involved in the lysine biosynthetic pathway. The product of this pathway is precursors of peptidoglycan synthesis, which is a component of bacterial cell wall. Also, because mammals lack this enzyme, consequently E. faecalis diaminopimelate epimerase (EfDapF) represents a potential target for developing novel class of antibiotics. In this regard, we have successfully cloned, overexpressed the gene encoding DapF in BL‐21(DE3) and purified with Ni‐NTA Agarose resin. In addition to this, binding studies were performed using fluorescence spectroscopy in order to confirm the bindings of the identified lead compounds (acetaminophen and dexamethasone) with EfDapF. Docking studies revealed that acetaminophen found to make hydrogen bonds with Asn72 and Asn13 while dexamethasone interacted by forming hydrogen bonds with Asn205 and Glu223. Thus, biochemical studies indicated acetaminophen and dexamethasone, as potential inhibitors of EfDapF and eventually can reduce the catalytic activity of EfDapF.

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Dimerization of Bacterial Diaminopimelate Epimerase Is Essential for Catalysis

Cited by 43 publications

References 53 publications

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Dimerization of Bacterial Diaminopimelate Decarboxylase Is Essential for Catalysis

Through the Looking Glass: Chiral Recognition of Substrates and Products at the Active Sites of Racemases and Epimerases

In silico prediction, molecular docking and binding studies of acetaminophen and dexamethasone to Enterococcus faecalis diaminopimelate epimerase

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