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Organophosphonate-Degrading Phnz Reveals an Emerging Family of HD Domain Mixed-Valent Diiron Oxygenases

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Organophosphonate-Degrading Phnz Reveals an Emerging Family of HD Domain Mixed-Valent Diiron Oxygenases Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases Bigna Wörsdörfera,1, Mahesh Lingarajub, Neela H. Yennawarc, Amie K. Boala,b, Carsten Krebsa,b, J. Martin Bollinger, Jr.a,b,1, and Maria-Eirini Pandeliaa,1 Departments of aChemistry, and bBiochemistry and Molecular Biology, and cHuck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802 Edited by Edward I. Solomon, Stanford University, Stanford, CA, and approved October 11, 2013 (received for review August 22, 2013) The founding members of the HD-domain protein superfamily are the conserved H...HD...H...H...D motif binds two iron ions, phosphohydrolases, and newly discovered members are generally and the enzyme uses an unprecedented mixed-valent FeII/FeIII – annotated as such. However, myo-inositol oxygenase (MIOX) exem- cofactor form to catalyze the four-electron (4e ) oxidative C-C plifies a second, very different function that has evolved within the bond cleavage converting myo-inositol (MI; cyclohexan-1,2,3,5/4, III common scaffold of this superfamily. A recently discovered HD pro- 6-hexa-ol) to D-glucuronate (13, 16, 17). The Fe site coor- tein, PhnZ, catalyzes conversion of 2-amino-1-hydroxyethylphospho- dinates MI, serving as Lewis acid to activate the substrate, and II III III nate to glycine and phosphate, culminating a bacterial pathway for the Fe site activates O2 to produce a superoxo-Fe /Fe in- the utilization of environmentally abundant 2-aminoethylphosph- termediate that initiates the reaction by abstracting a hydrogen atom from C1 of MI (6, 18). onate. Using Mössbauer and EPR spectroscopies, X-ray crystallog- – raphy, and activity measurements, we show here that, like MIOX, The recently discovered HD protein, PhnZ, catalyzes the 4e II III PhnZ employs a mixed-valent Fe /Fe cofactor for the O2-dependent oxidative C-P bond cleavage of 2-amino-1-hydroxyethylphos- oxidative cleavage of its substrate. Phylogenetic analysis suggests phonate (OH-AEP) to glycine and phosphate (19) (Fig. 1). It is that many more HD proteins may catalyze yet-unknown oxygen- the second of two enzymes in a recently discovered pathway BIOCHEMISTRY ation reactions using this hitherto exceptional FeII/FeIII cofactor. that degrades the most abundant environmental phosphonate The results demonstrate that the catalytic repertoire of the HD compound, 2-aminoethylphosphonate (2-AEP) (20–23), and its superfamily extends well beyond phosphohydrolysis and suggest reaction represents the third type of enzymatic C-P bond cleavage that the mechanism used by MIOX and PhnZ may be a common to be recognized (23–26). PhnZ was shown to require iron for strategy for oxidative C-X bond cleavage. activity, but the nature of the iron cofactor and its mechanism are unknown (19). The first enzyme in the pathway, the FeII- α α nonheme diiron enzymes | C-H activation | superoxo intermediate | PhnY | and -ketoglutarate( KG)-dependent oxygenase, PhnY, introduces structural genomics the hydroxyl group on C1 of 2-AEP, activating the compound for subsequent C-P bond cleavage by PhnZ (19). The assignment ith the number of known protein sequences rising expo- fi Wnentially, assignment of functions to the ever-expanding Signi cance proteome is a central, largely unmet challenge in the molecular life sciences. Assignment to a known superfamily based on se- Evolution functionally diversifies conserved protein architectures, quence or structure is usually the first approach to predict the precluding assignment of function from structure alone. The HD function of an uncharacterized protein and often provides structural domain was first recognized in a group of phospho- valuable hints for further studies. However, nature uses di- hydrolases and came to be associated with that activity, but vergent evolution to create new functions, leading to functional characterization of the archetypal mixed-valent diiron oxygen- diversification within superfamilies that can be as modest as ase (MVDO), myo-inositol oxygenase, attributed a very different different substrate specificities or as profound as fundamentally activity, O2-mediated C-C bond cleavage, to an HD protein. We different reaction types (1). Without any biochemical or bi- demonstrate that the recently discovered C-P bond-cleaving ological information, functional predictions based solely on su- enzyme, PhnZ, is another example of an HD-domain MVDO. perfamily assignment can thus be misleading or incorrect (2). Sequence and functional data for the dimetal HD proteins reveal The HD domain superfamily, recognized by Aravind and that they segregate into well-defined clades, of which several Koonin in 1998 on the basis of sequence analysis (3), now con- are more likely to comprise MVDOs than phosphohydrolases. tains more than 37,000 members occurring in a broad range of This study provides a basis to assign hydrolase or oxygenase organisms in all three domains of life and in more than 240 activity to proteins in this largely uncharacterized enzyme distinct domain architectures (4). HD proteins possess a con- superfamily. served α-helical core containing their characteristic H...HD...D II II sequence motif that binds a divalent metal ion (often Zn ,Mg , Author contributions: B.W., C.K., J.M.B., and M.-E.P. designed research; B.W. and M.-E.P. or MnII) (5). HD proteins with a dinuclear metal cluster, in performed research; M.L., N.H.Y., and A.K.B. crystallized PhnZ and solved the X-ray struc- which two additional histidines from within the D...D region of tures; M.L., N.H.Y., and A.K.B. contributed new reagents/analytic tools; B.W., C.K., and the domain form a second metal binding site, have also been M.-E.P. analyzed data; and B.W., J.M.B., and M.-E.P. wrote the paper. identified (6, 7). Only a limited number of HD proteins have The authors declare no conflict of interest. been biochemically characterized; they almost exclusively cata- This article is a PNAS Direct Submission. lyze phosphoester hydrolysis, which has led to the general as- Freely available online through the PNAS open access option. signment of HD proteins as phosphohydrolases (3, 5, 8–12). The Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, wide phylogenetic distribution and diverse domain architectures www.pdb.org (PDB ID codes 4N6W and 4N71). of the HD proteins suggest, however, that additional activities 1To whom correspondence may be addressed. E-mail: [email protected], jmb21@psu. might have evolved within the superfamily. Indeed, the enzyme edu, or [email protected]. myo-inositol oxygenase (MIOX) exemplifies a mechanistically very This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. different catalytic function for an HD protein (13–15). In MIOX, 1073/pnas.1315927110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1315927110 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 O (SI Appendix,TableS1), confirms that the diiron cluster PhnZ O O - + + detected in the Mössbauer spectra is coordinated by the resi- + P O H3N + P OH + H H3N - - dues in the H...HD...H...H...D sequence motif, as in other O O2 O- O - OH O structurally characterized HD proteins with dinuclear metal clusters [see, for example, Protein Data Bank (PDB) ID codes Fig. 1. Reaction catalyzed by PhnZ. 3TM8, 2IBN, 3CCG, 2O08, 2OGI, 3GW7, and 2PQ7 discussed below]. The coordination geometry of the PhnZ diiron cluster is nearly identical to the cluster geometries observed in these other of PhnZ to the HD domain superfamily, the conservation of the six proteins (Fig. 3). The enzyme is a monomer, and the core helices residues that coordinate the cofactor in MIOX, the dependence of near the active site superimpose well with those of MIOX its catalytic activity on the presence of iron, and the analogy (2.05 Å rmsd for 125 Cα atoms; ref. 30). However, PhnZ is smaller between the PhnZ-catalyzed oxidation and that catalyzed by (190 aa) than MIOX (285 aa), and the topology of peripheral MIOX all suggest that PhnZ might provide the second example structural elements differs significantly between the two proteins of an HD protein using a mixed-valent diiron cofactor to catalyze (SI Appendix,Fig.S3)(6).Thefive α-helices characteristic of an oxygenation (rather than a hydrolysis) reaction. the HD scaffold contribute the six conserved iron ligands (His34, Here, we demonstrate by Mössbauer and EPR spectroscopy His58, Asp59, His80, His104, and Asp161). The iron ions, which that PhnZ harbors a diiron cluster and, like MIOX, markedly II III are modeled at almost full occupancy (0.92), are most likely both stabilizes the mixed-valent Fe /Fe state. The X-ray crystal III fi in the Fe oxidation state and are separated by 3.7 Å, similar to structure of PhnZ con rms the presence of a diiron cofactor and the distance seen in the structures of MIOX from Mus musculus shows that the substrate is bound to the site 2 Fe ion in a manner and Homo sapiens (3.65 Å and 3.8 Å; PDB ID codes 2HUO and analogous to the binding mode of MI in MIOX. PhnZ is an 2IBN) (6, 31). The two Fe sites are bridged by the aspartate of oxygenase requiring O2 for its reaction, as determined by activity the HD motif and one μ-oxo/hydroxo bridge, as also seen in the assays and isotope-tracer experiments. Freeze-quench (FQ) EPR MIOX structures (Fig. 3 B and C). Citrate from the crystalli- experiments demonstrate that the FeII/FeIII state is competent to − zation solution binds to the active site with well-defined electron react rapidly with O2 to effect the 4e oxidation of OH-AEP. Our density (SI Appendix,Fig.S5). Its hydroxyl group and Oβ1ofthe results thus confirm that PhnZ is the second example of an HD fi II III central carboxyl group coordinate to the Fe2 ion, forming a ve- protein using a Fe /Fe diiron cofactor for oxidative bond membered ring.
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