The dapE-Encoded N-Succinyl-L, L-Diaminopimelic Acid Desuccinylase from Haemophilus influenzae Contains two Active Site Histidine Residues

Department of Chemistry, Loyola University Chicago, 1068 W Sheridan Rd, Chicago, IL 60626, USA.
European Journal of Biochemistry (Impact Factor: 2.54). 09/2008; 14(1):1-10. DOI: 10.1007/s00775-008-0418-z
Source: PubMed


The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 A of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical.

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Available from: Boguslaw Nocek, Sep 29, 2015
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    • "Futhermore, studies involving Zn K-edge extended X-ray absorption fine structure (EXAFS) analyses of H. influenzae SDAP-DS indicate that the enzyme contains dinuclear Zn 2+ active sites (Cosper et al., 2003). Studies of H. influenzae SDAP-DS mutants by kinetics, electronic absorption spectroscopy and electron paramagnetic resonance spectroscopy showed that His67 and His349 coordinate Zn 2+ ions, with His67 functioning in catalysis (Gillner et al., 2009). A similar study showed that residue Glu134 is also involved in catalysis, possibly functioning as an acid/base (Davis et al., 2006). "
    Biochemistry, 03/2012; , ISBN: 978-953-51-0076-8
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    ABSTRACT: The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase (DapE) is a critical bacterial enzyme for the construction of the bacterial cell wall. A screen biased toward compounds containing zinc-binding groups (ZBG's) including thiols, carboxylic acids, boronic acids, phosphonates and hydroxamates has delivered a number of micromolar inhibitors of DapE from Haemophilus influenzae, including the low micromolar inhibitor L-captopril (IC(50)=3.3 microM, K(i)=1.8 microM). In vitro antimicrobial activity was demonstrated for L-captopril against Escherichia coli.
    Bioorganic & medicinal chemistry letters 09/2009; 19(22):6350-2. DOI:10.1016/j.bmcl.2009.09.077 · 2.42 Impact Factor
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    ABSTRACT: Biosynthesis of lysine and meso-diaminopimelic acid in bacteria provides essential components for protein synthesis and construction of the bacterial peptidoglycan cell wall. The dapE operon enzymes synthesize both meso-diaminopimelic acid and lysine and, therefore, represent potential targets for novel antibacterials. The dapE-encoded N-succinyl-L,L-diaminopimelic acid desuccinylase functions in a late step of the pathway and converts N-succinyl-L,L-diaminopimelic acid to L,L-diaminopimelic acid and succinate. Deletion of the dapE gene is lethal to Helicobacter pylori and Mycobacterium smegmatis, indicating that DapE's are essential for cell growth and proliferation. Since there are no similar pathways in humans, inhibitors that target DapE may have selective toxicity against only bacteria. A major limitation in developing antimicrobial agents that target DapE has been the lack of structural information. Herein, we report the high-resolution X-ray crystal structures of the DapE from Haemophilus influenzae with one and two zinc ions bound in the active site, respectively. These two forms show different activity. Based on these newly determined structures, we propose a revised catalytic mechanism of peptide bond cleavage by DapE enzymes. These structures provide important insight into catalytic mechanism of DapE enzymes as well as a structural foundation that is critical for the rational design of DapE inhibitors.
    Journal of Molecular Biology 02/2010; 397(3):617-26. DOI:10.1016/j.jmb.2010.01.062 · 4.33 Impact Factor
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