Characterization of Metal Binding in the Active Sites of Acireductone Dioxygenase Isoforms from Klebsiella ATCC 8724

Abstract

The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities towards their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M +2 metal ion bound in the active site. The Ni +2 -bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate and carbon monoxide, while the Fe +2 -bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS) and isothermal calorimetry measurements, it is concluded that the same four residues, His 96, His 98, Glu 102 and His 140, provide the protein-based ligands for the metal in both the Ni-and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a His ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved non-ligand acidic residues, Glu 95 and Glu 100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from in vivo sources or prevent the loss of metal to stronger chelators. The Glu 100 mutant reconstitutes readily but has low activity. Mutation of Asp 101 results in an active enzyme that incorporates metal in vivo but shows evidence of mixed forms.* To whom correspondence should be addressed: pochapsk@brandeis.edu, phone 781−736−2559, fax 781−736−2516. e Current address: Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University, 70 Ship Street, GE3, Providence, RI, 02912 f Current address: Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, 635 Barnhill Dr., Room 1007, Indianapolis IN 46202 g This work was supported in part by USPHS grant (R01-GM067786 TCP). Acknowledgment is made to the donors of The American Chemical Society Petroleum Research Fund for partial support (MJM). Trainee funding (SCC) was provided by the NIH-Chemistry Biology Interface Program, T32-GM08515. XAS data collection at the National Synchrotron Light Source at Brookhaven National Laboratory was supported by the U.S. Department of Energy, Division of Materials Sciences and Division of Chemical Sciences. Beamline X9B at NSLS is supported in part by the NIH.Supporting information available Fits for resting state and ES...

Metal and Ligand Control on Structure and Functions of Ni(Fe)ARD: Role of Supramolecular Structures

“…(before Ni(Fe)ARD activity was discovered by Pochapsky et al [1,12] for bacteria and plants). Interestingly, in the case of KoARD (Klebsiella oxytoca ARD, bacterial enzyme ARD) unlike MmARD, Ni-bound form has higer activity than Fe-KoARD [1].…”

“…Specific structural organization of iron complexes may facilitate the following regioselective addition of activated oxygen to Acireductone ligand and the reactions leading to formation of methionine (and ethylene also). So, in the Unlike catalysis with Fe-ARD, mechanism of catalysis by Ni-ARD does not include O 2 -activation, and oxygenation of Acireductone leads to the formation of products not being precursors of methionine [1,11,12]. In our previous works, we have shown that formation of multidimensional forms based on nickel complexes can be one of the ways of regulating the activity of two enzymes [4].…”

“…Namely, carbon monoxide, CO, is formed because of action of nickel-containing Dioxygenase Ni-ARD. It was established, that CO is a representative of the new class of neural messengers, and seems to be a signal transducer like nitrogen oxide, NO [1,11,12]. earlier with AFM method.…”

Acireductone Dioxygenase 1 (ARD1) Is an Effector of the Heterotrimeric G Protein β Subunit in Arabidopsis

“…A 5 ppb indium solution (in 2% HNO 3 ) was used as the instrument internal standard during the runs. Isotopes 24 Mg, 55 Mn, 57 Fe, 59 Co, and 60 Ni were quantitatively monitored in a peak hopping mode with a total of 100 scans per point per isotope (50,000 s of dwell time and five replicates of 20 scans).…”

Dual chemistry catalyzed by human acireductone dioxygenase

“…Both these forms of ARD have been isolated from native K. oxytoca (Myers et al, 1993), and are also obtained upon overexpression in Escherichia coli. The activities of the two enzymes can be interconverted by replacement and reconstitution with the appropriate metal (Chai et al, 2008). These represent the only known pair of naturally occurring metalloenzymes with distinct functions determined solely by the metal ion identity in the active site.…”