Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

Wörsdörfer, Bigna; Lingaraju, Mahesh; Yennawar, Neela H.; Boal, Amie K.; Krebs, Carsten; Bollinger, J. Martin; Pandelia, Maria-Eirini

doi:10.1073/pnas.1315927110

Cited by 51 publications

(128 citation statements)

References 45 publications

(60 reference statements)

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“…During review of this manuscript Wörsdörfer et al reported elegant Mössbauer and EPR spectroscopic data confirming that PhnZ uses a mixed-valence di-iron active site for catalysis (30). They also reported a low-resolution X-ray crystal structure of PhnZ bound to (R)-2 together with a citrate bound structure.…”

Section: Discussionmentioning

confidence: 92%

Crystal structure of PhnZ in complex with substrate reveals a di-iron oxygenase mechanism for catabolism of organophosphonates

Staalduinen

McSorley

Schiessl

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

118

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Significance Inorganic phosphate (Pi) is an essential component of many biological molecules and thus is required by all life forms. However, soluble Pi is typically at low abundance in the environment. To compensate, microbes have evolved unique carbon–phosphorus-bond cleaving reactions to use organophosphonates as an alternative source of Pi. The marine-derived enzyme PhnZ utilizes a new oxidative mechanism for CP bond cleavage involving iron and molecular oxygen. The three-dimensional structure of PhnZ reveals unique active site features that contribute to catalysis of CP bond cleavage and substrate specificity, as well as an evolutionary link between phosphodiester bond hydrolysis and oxidative bond cleavage. This evolutionary link likely reflects the ancient origins of organophosphonates in the environment.

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Section: Discussionmentioning

confidence: 92%

Crystal structure of PhnZ in complex with substrate reveals a di-iron oxygenase mechanism for catabolism of organophosphonates

Staalduinen

McSorley

Schiessl

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

118

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“…4A). The two metal centers are separated by 5.3-and 5.2-Å for the OxsA OXT-PP and OxsA OXT-PPP structures, respectively, which is different from other HD domain dinuclear sites, which are separated by 3.4-3.8 Å[PDB ID codes 3TM8 (11), 2IBN (29), 3CCG 2O08, 2OGI, 2PQ7, and 4N6W (18)] and bridged by the HD doublet Asp residue (Fig. 4 E-G).…”

Section: +mentioning

confidence: 99%

“…This difference could be due in part to the fact that the commercially available deoxyribonucleotide compounds are less pure (98-100%, ≥95%, The Fe ions in PhnZ are 3.7 Å apart, bridged by a μ-oxo moiety and a citrate molecule from the crystallization buffer that binds in place of substrate (18). The second Fe site has three protein ligands (18). In A-C, the proposed nucleophilic water is indicated by an asterisk (*).…”

Section: +mentioning

confidence: 99%

“…This superfamily is divided into classes of enzymes that use a His-Asp doublet of residues and an additional series of conserved His and Asp residues to coordinate a single divalent metal (HX n HDX n D motif) (8,9), a dinuclear metal active site (HX n HDX n HX n HX n D motif) (10,11), or a recently described trinuclear iron metal-center (12). Although in many cases the identity of the relevant catalytic metal required for chemistry in vivo is unclear, there are examples of mononuclear enzymes that can use magnesium (13), cobalt (14), or manganese (8,13,15), and dinuclear enzymes that can use nickel (16), manganese (17), or iron (10,(18)(19)(20).…”

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confidence: 99%

“…Finally, two HD domain enzymes, myo-inositol oxygenase and PhnZ, are not hydrolases at all. They bind mixedvalent Fe 2+ /Fe 3+ metal centers and operate as oxygenases, indicating the chemical diversity possible with the HD domain fold (10,18,20,(24)(25)(26).…”

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confidence: 99%

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An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis

Bridwell‐Rabb

Kang

Zhong

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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HD domain phosphohydrolase enzymes are characterized by a conserved set of histidine and aspartate residues that coordinate an active site metallocenter. Despite the important roles these enzymes play in nucleotide metabolism and signal transduction, few have been both biochemically and structurally characterized. Here, we present X-ray crystal structures and biochemical characterization of the Bacillus megaterium HD domain phosphohydrolase OxsA, involved in the biosynthesis of the antitumor, antiviral, and antibacterial compound oxetanocin-A. These studies reveal a previously uncharacterized reaction for this family; OxsA catalyzes the conversion of a triphosphorylated compound into a nucleoside, releasing one molecule of inorganic phosphate at a time. Remarkably, this functionality is a result of the OxsA active site, which based on structural and kinetic analyses has been tailored to bind the small, four-membered ring of oxetanocin-A over larger substrates. Furthermore, our OxsA structures show an active site that switches from a dinuclear to a mononuclear metal center as phosphates are eliminated from substrate.X-ray crystallography | phosphohydrolase | metalloenzymes | natural products | nucleosides

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The Di‐Iron OxygenasePhnZ

Séguin

Zechel

2016

Encyclopedia of Inorganic and Bioinorganic Chemistry

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PhnZ is a di‐iron‐dependent monooxygenase from the HD superfamily of metal ion‐dependent phosphohydrolases. These enzymes utilize a conserved histidine‐aspartate motif in the active site for metal ion binding. PhnZ is commonly found in marine bacteria where it works in tandem with the α‐ketoglutarate‐dependent dioxygenase PhnY to extract inorganic phosphate from the organophosphonate natural product 2‐aminoethylphosphonic acid. PhnY hydroxylates 2‐aminoethylphosphonic acid stereospecifically to generate ( R )‐2‐amino‐1‐hydroxyethylphosphonic acid, whereupon PhnZ catalyzes the oxidative cleavage of the carbon–phosphorus bond yielding glycine and inorganic phosphate. Analysis of the metal ion dependence of PhnZ through activity assays, ICP‐MS, Mössbauer spectroscopy, and EPR spectroscopy have shown that this enzyme utilizes a mixed‐valence Fe(II)/Fe(III) di‐iron cofactor for catalysis. The X‐ray crystal structures of PhnZ have revealed that one Fe ion (Fe2) is used to bind the substrate in a bidentate manner, while the other Fe ion (Fe1) is likely used to reduce dioxygen. Two conserved active site residues, Y24 and E27, are observed to migrate in and out of the active site in response to substrate binding. Y24 is observed to bind Fe2, occupying the putative dioxygen binding site, but is expelled upon recognition of the substrate amino group by the residue E27. This is proposed to allow dioxygen to bind to Fe1, placing it well within proximity of the substrate bound at Fe2. A mechanism for oxidative carbon–phosphorus bond cleavage by PhnZ has been proposed based on these results. PhnZ not only represents the third known mechanism for enzymatic cleavage of CP‐bonds but also provides a window into the evolution of new functions within an enzyme superfamily.

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Organophosphonate-degrading PhnZ reveals an emerging family of HD domain mixed-valent diiron oxygenases

Cited by 51 publications

References 45 publications

Crystal structure of PhnZ in complex with substrate reveals a di-iron oxygenase mechanism for catabolism of organophosphonates

Crystal structure of PhnZ in complex with substrate reveals a di-iron oxygenase mechanism for catabolism of organophosphonates

An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis

The Di‐Iron OxygenasePhnZ

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