J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 DOI 10.1007/s00775-009-0515-7 POSTER PRESENTATION Metalloproteins P501 1Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Mechanisms of isoniazid activation inferred Germany, 2Department of Chemistry, Bielefeld University, 33615 Bielefeld, from resistance mutation catalase-peroxidases (KatGs) Germany, 1 1 Christine E. Fairchild , Reza A. Ghiladi 3EMBL Hamburg, 22603 Hamburg, Germany, 1 Department of Chemistry, NC State University, Raleigh, 4Max Planck Institute of Biophysics, 60438 Frankfurt am Main, NC 27607, USA. [email protected] Germany. [email protected] Mycobacterium tuberculosis catalase-peroxidase (KatG) activates the [Fe]-hydrogenase is one of three types of enzymes known to activate pro-drug isoniazid (INH) to the isonicotinoyl acyl radical, which then molecular hydrogen (H2). It catalyzes the generation of a hydride that combines with NADH, forming an INH–NADH adduct capable of is required to reduce methenyltetrahydromethanopterin (methenyl- + inhibiting InhA, an enzyme involved in cell wall biosynthesis. H4MPT ) to methylene-H4MPT, which is an intermediary step of Inability to form mycolic acid, which comprises a protective layer of methanogenesis in many methanogenic archaea. Recently, the crystal the cell wall, causes the observed bactericidal activity. However, structure of its reconstituted holoenzyme was solved and revealed the drug-resistant strains of tuberculosis (TB) can arise from mutations in novel structure of the mononuclear iron complex in the active site. KatG. Thus, before novel drug therapies can be developed, a new We report here on the structure of the C176A mutated holoenzyme, understanding of the underlying causes of drug resistance is needed. which was crystallized in the presence of dithiothreitol (DTT) Over 25 different resistance mutation KatGs have been expressed, (Fig. 1). The active site iron center of the enzyme is octahedrally purified to, or near, homogeneity, and characterized for catalase and coordinated by one DTT sulfur, one DTT oxygen, two carbon mon- peroxidase activities as well as their ability to form the INH–NADH oxide ligands, the pyridinol nitrogen and the pyridinol formylmethyl adduct using the following: peroxide (tBuOOH and the hydrogen carbon in acyl-iron ligation (Fig. 2). This result prompted us to peroxide generating enzymatic system glucose/glucose oxidase), reinterpret the iron complex structure in the wild-type enzyme and superoxide (xanthine/xanthine oxidase), and no exogenous oxidant. would suggest an alternative open coordination site for H2 binding. No correlation was found between adduct formation and either cat- alase or peroxidase activity. Mutated residue location plays a role in available pathways of INH activation and causes of resistance. These results as well as further characterization with stopped-flow UV-vis- ible spectroscopy will be presented. Fig. 1 Overall structure Fig. 1 Resistance mutations studied in KatG P502 The crystal structure of C176A mutated [Fe]-hydrogenase suggests an acyl-iron ligation in the active site iron complex Takeshi Hiromoto1, Kenichi Ataka2, Oliver Pilak1, Sonja Vogt1, Marco Salomone Stagni3, Wolfram Meyer-Klaucke3, Eberhard Warkentin4, Rudolf K. Thauer1, Ulrich Ermler4, Seigo Shima1 Fig. 2 Active site iron complex 123 S186 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 P503 1School of Chemistry and Molecular Biosciences, The University of Substrate and metal ion modulate the catalytic Queensland, Brisbane 4072, Australia, 2Research School Of Chemistry, The Australian National University, mechanism of GpdQ: a promising bioremediator Canberra 0200, Australia. [email protected] for organophosphate nerve agents and pesticides. The organophosphate-degrading enzyme from Agrobacterium tum- Kieran S. Hadler1, Fernanda Ely1, Natasa Mitic1, efaciens (OPDA) is a metallohydrolase which is able to degrade Lawrence R. Gahan1, David L. Ollis2, James A. Larrabee3, highly toxic organophosphate pesticides and nerve gas agents into Gerhard Schenk1 less or non-toxic compounds. The reaction mechanism employed 1School of Chemistry and Molecular Biosciences, The University of by OPDA is not yet fully understood, but there are two currently Queensland, St Lucia 4072, Australia, accepted hypotheses involving either (a) a metal ion-bridging or (b) 2Research School of Chemistry, Australian National University, a terminally coordinated hydroxide/water nucleophile. In this work, Canberra 0200, Australia, we have investigated the role of the coordinated hydroxide/water 3Department of Chemistry and Biochemistry, Middlebury College, molecules in the nucleophilic reaction. The pH dependence of the Middlebury 05753, USA. [email protected] kinetic properties of several metal ion derivatives of wild type The glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes OPDA, including Co(II), Zn(II) and Cd(II), yielded catalytically is a promiscuous binuclear metallophosphatase with a remarkable relevant pKa values that implicate a metal ion-bridging water ability to hydrolyze all classes of phosphate ester substrates. Notably, molecule in the reaction mechanism. The Co(II) derivative, how- it is capable of degrading the toxic byproducts of the hydrolysis of ever, has shown a second relevant protonation equilibrium (pKa VX (a powerful nerve agent) and a range of organophosphate pesti- 10.1), which is ascribed to a terminally metal ion-bound water. The cides (e.g. paraoxon and demeton). Therefore, GpdQ is of interest for 1.65 A˚ resolution structure of OPDA, crystallised in presence of its potential application as versatile enzymatic bioremediator [1]. the very slow substrate diethyl 4-methoxyphenyl phosphate (EPO) Recently, we have shown that GpdQ employs an unusual catalytic facilitated the capture of an OPDA structure with both bound mechanism where the formation of the catalytically competent substrate and product present in the active site. Preliminary results binuclear enzyme is induced by substrate and is regulated by the indicate that upon substrate binding, the bridging hydroxide, which coordination flexibility of an asparagine ligand [2]. In order to is also present in the free enzyme, is displaced to a terminally investigate the individual stages of this mechanism, stopped flow metal ion-bound position, where it is favourably placed to carry out fluorescence has been utilized, looking specifically at the pre-steady a nucleophilic attack on the substrate phosphorous atom. In addi- state features. In addition, the steady-state kinetic behaviour for a tion, kinetic analyses, as well as the crystal structures of the Y257F number of metal-derivatives of GpdQ has been measured in order to and R254H mutants have demonstrated the significance of a understand the role of the metal ions in catalysis. hydrogen-bonding network in the second coordination sphere in orienting the substrate for hydrolysis. P505 Structural and catalytic models for phosphoesterases Lawrence R. Gahan1, Gary Schenk1, David Ollis2 1School of Chemistry and Molecular BioSciences, The University of Queensland, St Lucia, QLD 4072, Australia, 2Research School of Chemistry, The Australian National University, Canberra, ACT, Australia. [email protected] The prolonged and widespread agricultural application of organo- phosphate pesticides (OPs) has contributed to increased agricultural production and to environmental problems related to run-off and subsequent contamination of water sources. OP-degrading com- pounds are of interest therefore for environmental detoxification, References but also as agents for protection against bioterrorism; nerve gas 1. Ghanem E, Li Y, Xu C, Raushel FM (2007) Biochemistry agents VX and sarin are both OPs. Our interest is in models for the 46:9032 active sites of metallohydrolases, including the purple acid phos- 2. Hadler KS, Tanifum EA, Yip SH-C, Mitic´ N, Guddat LW, phatases (PAP), the glycerophosphodiester degrading enzyme from Jackson CJ, Gahan LR, Nguyen K, Carr PD, Ollis DL, Hengge Enterobacter aerogenes (GpdQ) and the structurally related OP- AC, Larrabee JA, Schenk G (2008) J Am Chem Soc 130:14129 degrading triesterase from Agrobacterium radiobacter (OpdA). Here, we focus on GpdQ, a universal phosphoesterase, shown to have activity towards phosphomono-, di- and tri-esters (including OPs). GpdQ shares six of seven donor ligands with PAP (Figure), P504 the notable difference being the lack of a tyrosine ligand. GpdQ Catalytic mechanism of the reaction catalyzed also shares five donor ligands with OpdA, with the metal centers coordinated by four histidines, an aspartate and a lysine. The native by the binuclear organophosphate-degrading enzyme metal ion composition of GpdQ is still unknown, but enzymatic from Agrobacterium tumefaciens activity can be reconstituted with Zn2+,Co2+ and Mn2+. We report Fernanda Ely1, Paul Carr2, Luke Guddat1, David Ollis2, the synthesis and characterization of new biomimetic systems as Lawrence Gahan1, Gary Schenk1 structural models for GpdQ. 123 J Biol Inorg Chem (2009) 14 (Suppl 1):S185–S224 S187 osteoporosis [2]. Therefore, a thorough understanding of the mecha- nism is crucial. Our aim on one hand is to find a suitable model system to mimic the active site of PAP. We have developed a new ligand based on cyclam with two distinct coordination sites that may form (hydr)oxo-bridged dinuclear complexes. The coordination chemistry of this ligand is illustrated. H N N N N N Acknowledgements: This work was funded by grants from the Aus- N tralian Research Council