The Zn K-edge extended X-ray absorption fine structure (EXAFS) spectra, of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae have been recorded in the presence of one or two equivalents of Zn(II) (i.e. [Zn_(DapE)] and [ZnZn(DapE)]). The Fourier transforms of the Zn EXAFS are dominated by a peak at ca. 2.0 Å, which can be fit for both [Zn_(DapE)] and [ZnZn(DapE)], assuming ca. 5 (N,O) scatterers at 1.96 and 1.98 Å, respectively. A second-shell feature at ca. 3.34 Å appears in the [ZnZn(DapE)] EXAFS spectrum but is significantly diminished in [Zn_(DapE)]. These data show that DapE contains a dinuclear Zn(II) active site. Since no X-ray crystallographic data are available for any DapE enzyme, these data provide the first glimpse at the active site of DapE enzymes. In addition, the EXAFS data for DapE incubated with two competitive inhibitors, 2-carboxyethylphosphonic acid and 5-mercaptopentanoic acid, are also presented

Bienvenue, David L.

Cosper, Nathaniel J.

Gilner, Danuta M.

Holz, Richard C.

Scott, Robert A.

Shokes, Jacob E.

Tsukamoto, Takashi

English

epublications@Marquette

NOT THE PUBLISHED VERSION; this is the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Journal of the American Chemical Society, Vol 125, No. 48 (December 3, 2003): pg. 14654-14655. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. 1    The dapE-encoded N-Succinyl-l,l-Diaminopimelic Acid Desuccinylase from Haemophilus influenzae Is a Dinuclear Metallohydrolase   Nathaniel J. Cosper Department of Chemistry, University of Georgia, Athens, GA David L. Bienvenue Department of Chemistry and Biochemistry, Utah State University, Logan, UT Jacob E. Shokes Department of Chemistry, University of Georgia, Athens, GA Danuta M. Gilner Department of Chemistry and Biochemistry, Utah State University, Logan, UT Takashi Tsukamoto Guilford Pharmaceuticals Inc., Baltimore, MD NOT THE PUBLISHED VERSION; this is the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Journal of the American Chemical Society, Vol 125, No. 48 (December 3, 2003): pg. 14654-14655. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. 2  Robert A. Scott Department of Chemistry, University of Georgia, Athens, GA Richard C. Holz Department of Chemistry and Biochemistry, Utah State University, Logan, UT    Abstract  The Zn K-edge extended X-ray absorption fine structure (EXAFS) spectra, of the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae have been recorded in the presence of one or two equivalents of Zn(II) (i.e. [Zn_(DapE)] and [ZnZn(DapE)]). The Fourier transforms of the Zn EXAFS are dominated by a peak at ca. 2.0 Å, which can be fit for both [Zn_(DapE)] and [ZnZn(DapE)], assuming ca. 5 (N,O) scatterers at 1.96 and 1.98 Å, respectively. A second-shell feature at ca. 3.34 Å appears in the [ZnZn(DapE)] EXAFS spectrum but is significantly diminished in [Zn_(DapE)]. These data show that DapE contains a dinuclear Zn(II) active site. Since no X-ray crystallographic data are available for any DapE enzyme, these data provide the first glimpse at the active site of DapE enzymes. In addition, the EXAFS data for DapE incubated with two competitive inhibitors, NOT THE PUBLISHED VERSION; this is the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Journal of the American Chemical Society, Vol 125, No. 48 (December 3, 2003): pg. 14654-14655. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. 3  2-carboxyethylphosphonic acid and 5-mercaptopentanoic acid, are also presented.  According to recent estimates from the United States Centers for Disease Control and Prevention (the CDC), more than 10 million people died worldwide from bacterial infections in 1995.1 Tuberculosis, for example, was the leading cause of death in adults by an infectious disease.2 The importance of developing new drugs to fight infectious disease, such as tuberculosis, is underscored by the emergence of several pathogenic bacterial strains that are resistant to all currently available antibiotics.3-6 To overcome bacterial resistance to antibiotics, new enzyme targets must be located and small-molecule inhibitors developed. The meso-diaminopimelate (mDAP)/lysine biosynthetic pathway offers several potential anti-bacterial targets that have yet to be explored.7-9 Since both products of this pathway, mDAP and lysine, are essential components of the peptidoglycan cell wall in Gram-negative and some Gram-positive bacteria, inhibitors of enzymes within this pathway may provide a new class of antibiotics.5 The fact that there are no similar pathways in mammals suggests that inhibitors of enzymes in the mDAP/lysine pathway will provide selective toxicity against bacteria and will potentially have little or no effect on humans.   One of the enzymes in this pathway,10 the dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE), catalyzes the hydrolysis of N-succinyl-l,l-diaminopimelate to l,l-diaminopimelate and succinate.11 It has been shown that deletion of the gene encoding DapE is lethal to Helicobacter pylori and Mycobacterium smegmatis.12,13 Even in the presence of lysine-supplemented media, the DapE deletion cell line of H. pylori was unable to grow. Therefore, DapEs are essential for cell growth and proliferation. Since no structural data of any kind are available for DapE enzymes, we have recorded the Zn K-edge extended X-ray absorption fine structure (EXAFS) spectra of the DapE from H. influenzae in the presence of one or two equivalents of Zn(II) (i.e. [Zn_(DapE)] and [ZnZn(DapE)]) (Figure 1). For [ZnZn(DapE)] the EXAFS data report an average of both metal ion environments. The Fourier transforms of both [Zn_(DapE)] and [ZnZn(DapE)] are dominated by a peak at ca. 2.0 Å, that can be fit for both [Zn_(DapE)] and [ZnZn(DapE)] assuming 5 (N,O) scatterers, at 1.96 and 1.98 Å, respectively (Fits 1,3; Table 1). A NOT THE PUBLISHED VERSION; this is the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Journal of the American Chemical Society, Vol 125, No. 48 (December 3, 2003): pg. 14654-14655. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. 4  second shell feature at ca. 3.3 Å appears in the [ZnZn(DapE)] EXAFS spectrum but is significantly diminished in [Zn_(DapE)]. Fits incorporating a Zn−Zn interaction at 3.34 Å for [ZnZn(DapE)] have reasonable Debye−Waller factors (Fit 4; Table 1) and resulted in a significant improvement in goodness-of-fit values (f ‘). Therefore, the second-shell FT feature observed at 3.34 Å is consistent with a dinuclear Zn(II) active site in DapE. Although fits for [Zn_(DapE)] that include a Zn−Zn interaction improve the f ‘ values slightly, the Debye−Waller factor value for this interaction is significantly higher than for the same interaction in [ZnZn(DapE)] (cf. Fits 2,4; Table 1).    Figure 1 Zn K-edge X-ray absorption spectra for [Zn_(DapE)] (dashed) and [ZnZn(DapE)] (solid). (A) Zn edge spectra; (B) k3-weighted Zn EXAFS; (C) Fourier transforms over k = 2−13 Å-1, with S phase correction. NOT THE PUBLISHED VERSION; this is the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Journal of the American Chemical Society, Vol 125, No. 48 (December 3, 2003): pg. 14654-14655. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. 5   Figure 2 Zn K-edge X-ray absorption spectra of [ZnZn(DapE)] in the absence (black) and presence of CEPA (blue) or MSPA (red) and [ZnZn(APP)] in the presence of LPA (green). (A) Zn edge spectra; (B) k3-weighted Zn EXAFS; (C) Fourier transforms over k = 2−13 Å-1, with S phase correction. Table 1.  Curve-Fitting Results for Zn EXAFSa of DapE Enzymes sample filename (k range) Δk3χ fit shell Ras (Å) σas2 (Å2) ΔE0 (eV) f ‘ b  [Zn_(DapE)]  1  Zn−O5  1.96  0.0062  −9.11  0.104  ZDZ0A (2−13 Å-1)  2  Zn−O5  1.96  0.0062  −9.30  0.100  Δk3χ = 10.23    Zn−Zn  3.30  0.0117      [ZnZn(DapE)]  3  Zn−O5  1.98  0.0065  −9.01  0.111  ZDZ0A (2−13 Å-1)  4  Zn−O5  1.98  0.0064  −9.41  0.093  Δk3χ = 10.44    Zn−Zn  3.34  0.0051      [ZnZn(AAP)]  5  Zn−O5  1.99  0.0084  −8.71  0.094  ZAZZA (2−13 Å-1)  6  Zn−O5  1.99  0.0084  −9.12  0.089  Δk3χ = 9.47    Zn−Zn  3.27  0.0099      [ZnZn(DapE)]-CEPA  7  Zn−O5  1.99  0.0069  −8.00  0.096  ZDZ0A (2−13 Å-1)  8  Zn−O5  1.99  0.0085  −8.42  0.088  Δk3χ = 9.89    Zn−Zn  3.34  0.0083      [ZnZn(AAP)]-LPA  9  Zn−O5  1.99  0.0078  −8.22  0.108  ZAZPA (2−13 Å-1)  10  Zn−O5  1.99  0.0077  −8.56  0.103  Δk3χ = 9.91    Zn−Zn  3.30  0.0112        11  Zn−O5  1.99  0.0077  −8.49  0.103      Zn−Zn  3.80  0.0072      [ZnZn(DapE)]-MSPA  12  Zn−O5  1.99  0.0079  −3.03  0.109  ZDZTA (2−13 Å-1)  13  Zn−O5  1.99  0.0050  −6.24  0.097  Δk3χ = 9.98    Zn−S  2.30  0.0089          Zn−Zn  3.64  0.0083      NOT THE PUBLISHED VERSION; this is the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Journal of the American Chemical Society, Vol 125, No. 48 (December 3, 2003): pg. 14654-14655. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. 6  a  Shell is the chemical unit defined for the multiple scattering calculation, the number of scatterers per metal denoted by the subscript. Ras is the metal-scatterer distance. σas2 is a mean square deviation in Ras. ΔE0 is the shift in E0 for the theoretical scattering functions.b f ‘ is a normalized error (χ-squared):  f‘ = {∑i[k3(−)]2/N}1/2/[(k3χobs)max − (k3χobs)min]. Sequence alignments of several DapE genes with those of the crystallographically characterized aminopeptidase from Aeromonas proteolytica (AAP) and the carboxypeptidase G2 from Pseudomonas sp. strain RS-16 (CPG2), indicate that all of the amino acids that function as metal ligands in AAP and CPG2 are strictly conserved in DapEs.11,14-16 The X-ray crystal structures of both CPG2 and AAP reveal a (μ-aquo)(μ-carboxylato)dizinc(II) active site with one terminal carboxylate and histidine residue bound to each metal ion.15,16 Both Zn(II) ions in AAP and CPG2 reside in a distorted tetrahedral coordination geometry, with a Zn−Zn distance of 3.5 and 3.3 Å for AAP and CPG2, respectively.15,16 EXAFS data obtained for [ZnZn(AAP)] also contain a feature at 3.27 Å that can be modeled as a Zn−Zn vector (Fit 6; Table 1). Combination of these data indicates that DapE enzymes contain dinuclear Zn(II) active sites. In addition, on the basis of the first shell fits of [ZnZn(AAP)] and [ZnZn(DapE)], we propose that one carboxylate and one histidine residue resides at each Zn(II) site in DapE and that the two Zn(II) ions are bridged by one carboxylate residue and a water molecule.   The first step in inhibitor design for DapE enzymes requires an understanding of how inhibitors bind to the active site. Therefore, we recorded the Zn K-edge EXAFS spectra of [ZnZn(DapE)] in the presence of the competitive inhibitors 2-carboxyethylphosphonic acid (CEPA) and 5-mercaptopentanoic acid (MSPA) (Figure 2). The EXAFS data for [ZnZn(DapE)]−CEPA indicate that the average coordination number of each Zn(II) ion remains five and the Zn−Zn distance remains 3.34 (Fits 7,8; Table 1). An increase in the M−M distance of [ZnZn(AAP)], from 3.5 to 3.9 Å, is observed upon the addition of the transition-state analogue inhibitor l-leucine-phosphonic acid (LPA) which contains a similar ligating group to CEPA.17 The X-ray crystal structure and EXAFS data for [ZnZn(AAP)]-LPA, reveal that the bridging water molecule is displaced by LPA resulting in an η-1,2-μ-phosphonate bridge and an increase in the Zn−Zn distance of 0.4 Å (Fit 11; Table 1). The fact that the Zn−Zn distance does not change upon CEPA binding to [ZnZn(DapE)] suggests an η-1-μ-phosphonate NOT THE PUBLISHED VERSION; this is the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Journal of the American Chemical Society, Vol 125, No. 48 (December 3, 2003): pg. 14654-14655. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. 7  bridge exists, similar to the binding mode of LPA to the leucine aminopeptidase from bovine lens.17,18 On the other hand, MSPA binding to [ZnZn(DapE)] has a marked effect on the Zn K-edge, suggesting that the average electronic environment of the dinuclear Zn(II) site has changed significantly (Figure 2). The observed shift to lower energy in the absorption edge position is indicative of a net increase in electron density at the dinuclear Zn(II) site, consistent with a sulfur ligand. In addition, the EXAFS data for [ZnZn(DapE)]−MSPA reveal a new feature at 2.3 Å that is highly characteristic of a direct zinc−sulfur interaction (Fits 12,13; Table 1). Moreover, the M−M distance is lengthened from 3.34 to 3.64 Å. These data indicate that the thiol group of MSPA binds to one or more of the Zn(II) ions in the active site of DapE.   In conclusion, DapEs are potential molecular targets for a new and novel class of antibiotics. The EXAFS data presented herein provide the first structural information for any DapE enzyme and also establish the binding modes of phosphonate- and thiolate-containing inhibitors. In addition, the structural data obtained for CEPA bound to [ZnZn(DapE)] provide the first glimpse at the transition state of the hydrolysis reaction catalyzed by DapE. Since most pharmaceuticals target the transition state of enzymatic reactions, the structural aspects of [ZnZn(DapE)]−CEPA are particularly important for the rational design of new potent inhibitors of DapE enzymes.  Acknowledgment This work was supported by the National Institutes of Health (GM-42025, R.A.S.) and the National Science Foundation (CHE-0240810, R.C.H.). The E. coli BL21(DE3) strain expressing the recombinant H. influenzae dapE-encoded desuccinylase was provided by Professor John S. Blanchard (Albert Einstein College of Medicine, supported by AI-33696).  Supporting Information Available Experimental details (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.    NOT THE PUBLISHED VERSION; this is the author’s final, peer-reviewed manuscript. The published version may be accessed by following the link in the citation at the bottom of the page. Journal of the American Chemical Society, Vol 125, No. 48 (December 3, 2003): pg. 14654-14655. DOI. This article is © American Chemical Society and permission has been granted for this version to appear in e-Publications@Marquette. American Chemical Society does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from American Chemical Society. 8  References  1Henery, C. M. Chem. Eng. News 2000, 41−58. 2Snider, D. E.; Raviglione, M.; Kochi, A. In Global Burden of Tuberculosis; Bloom, B. R., Ed.; ASM Press:  Washington, DC, 1994; pp 3−11. 3Prevention. Centers for Disease Control and Prevention:  Washington, DC. MMWR Morb. Mortal. Wkly Rep. 1995, 44, 1−13. 4Howe, R. A.; Bowker, K. E.; Walsh, T. R.; Feest, T. G.; MacGowan, A. P. Lancet 1997, 351, 601−602. 5Levy, S. B. Sci. Am. 1998, 278, 46−53. 6Chin, J. New Sci. 1996, 152, 32−35. 7Scapin, G.; Blanchard, J. S. Adv. Enzymol. 1998, 72, 279−325. 8Born, T. L.; Blanchard, J. S. Curr. Opin. Chem. Biol. 1999, 3, 607−613. 9Girodeau, J.-M.; Agouridas, C.; Masson, M.; R., P.; LeGoffic, F. J. Med. Chem. 1986, 29, 1023−1030. 10Velasco, A. M.; Leguina, J. I.; Lazcano, A. J. Mol. Evol. 2002, 55, 445−459. 11Born, T. L.; Zheng, R.; Blanchard, J. S. Biochemistry 1998, 37, 10478−10487. 12Karita, M.; Etterbeek, M. L.; Forsyth, M. H.; Tummuru, M. R.; Blaser, M. J. Infect. Immun. 1997, 65, 4158−4164. 13Pavelka, M. S.; Jacobs, W. R. J. Bacteriol. 1996, 178, 6496−6507. 14Makarova, K. S.; Grishin, N. V. J. Mol. Biol. 1999, 292, 11−17. 15Chevrier, B.; Schalk, C.; D'Orchymont, H.; Rondeau, J.-M.; Moras, D.; Tarnus, C. Structure 1994, 2, 283−291. 16Rowsell, S.; Pauptit, R. A.; Tucker, A. D.; Melton, R. G.; Blow, D. M.; Brick, P. Structure 1997, 5, 337−347. 17Stamper, C.; Bennett, B.; Edwards, T.; Holz, R. C.; Ringe, D.; Petsko, G. Biochemistry 2001, 40, 7034−7046. 18Sträter, N.; Lipscomb, W. N. Biochemisty 1995, 34, 9200−9210.  

The \u3cem\u3edapE\u3c/em\u3e-encoded \u3cem\u3eN\u3c/em\u3e-Succinyl-l,l-Diaminopimelic Acid Desuccinylase from \u3cem\u3eHaemophilus influenzae\u3c/em\u3e Is a Dinuclear Metallohydrolase

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The \u3cem\u3edapE\u3c/em\u3e-encoded \u3cem\u3eN\u3c/em\u3e-Succinyl-l,l-Diaminopimelic Acid Desuccinylase from \u3cem\u3eHaemophilus influenzae\u3c/em\u3e Is a Dinuclear Metallohydrolase

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