# The HD-Domain Metalloprotein Superfamily:
### HD-Domain Oxygenases  
Most of the known HD-domain proteins are phosphohydrolases, but three members, namely _myo_-inositol oxygenase (MIOX), PhmZ, and TmpB, are monooxygenases and perform the  
Figure 5: Active site of HD-GYPs. HD-GYP proteins utilize an “H \(\ldots\) HD \(\ldots\) H \(\ldots\) HH \(\ldots\) D” motif that typically binds a dinuclear metal center. The third metal ion in the PmGH active site is stabilized by crystallization molecules shown in gray. In addition, these enzymes contain a GYP residue triad vicinal to the active site (shown in blue), the importance of which is currently unclear. Bd1817, which is inactive toward c-di-GMP, lacks the GYP tyrosine and the terminal aspartate is an asparagine. oxidative cleavage of a C-X bond [11; 12; 13; 15]. The discovery of this chemistry expands the catalytic repertoire of the HD-domain superfamily, and their conserved protein features may provide insight into the identification of yet-to-be-characterized HD-domain proteins as oxygenases. The first discovered HD-domain oxygenase, MIOX, catalyzes the oxidative cleavage of a C-C bond of _myo_-inositol to form D-glucuron acid (Figure 6) [11]. _Myo_-inositol is a precursor for inositol phosphoglycans, which act as insulin mediators, and altered inositol metabolism has been associated with diabetes. Therefore, the activity of MIOX is of increasing interest, as it presents a potential therapeutic target for treating both type-1 and type-2 diabetes. PhnZ and TmpB were later established as oxygenases, demonstrating that MIOX is not a functional outlier [12; 13]. Both PhnZ and TmpB are involved in the degradation of organophosphonates, which are compounds that serve as sources of inorganic phosphate for bacteria that occupy phosphate-limited environments (e.g., marine ecosystems) [12]. Unlike MIOX, PhnZ and TmpB act in tandem with the non-heme \(\alpha\)-ketoglutate (KG) dependent hydroxyles PhnY and TmpA, respectively, to cleave the C-P bond of their substrates (Figure 2) [12; 13]. PhnY initiates the degradation of 2-aminoethylphosphonate (2-AEP) via the addition of a hydroxyl group to the C1 carbon in a stereospecific manner producing (\(R\))-2-amino-1-hydroxyl phosphonate (OH-AEP) [12]. PhnZ performs the subsequent oxidative cleavage of the C-P bond of OH-AEP forming inorganic phosphate and glycine (Figures 2 and 6) [12]. The TmpA/TmpB pathway is mechanistically similar, with the only difference being the nature of the substrate, i.e., 2-(trimethylammonio)ethyl phosphonate (TMAEP) for TmpA [13]. MIOX, PhnZ, and TmpB bind a catalytically essential diiron cofactor via the HD-domain sequence \"H... HD... H... H... D\" (Figure 6). Each iron is coordinated in an octahedral geometry and bridged by the carboxylate group of the first aspartate residue in the HD-domain sequence as well as a \(\mu\)-oxo/hydroxo bridge (Figure 6) [15]. Unlike other dinuclear nonheme-iron oxygenases, which utilize the fully reduced Fe\({}^{\text{II}}\)/Fe\({}^{\text{III}}\) form of their cofactors, HD-domain oxygenases stabilize a mixed-valent Fe\({}^{\text{II}}\)/Fe\({}^{\text{III}}\) state for the four-electron oxidation of a C-C/P bond and do not require an external reducing system for reactivation of the cofactor (i.e., after one substrate turnover the site returns to the Fe\({}^{\text{II}}\)-Fe\({}^{\text{III}}\) form) [15]. Stabilization under the same redox conditions of the Fe\({}^{\text{II}}\)Fe\({}^{\text{III}}\) cofactor in oxygenases and the Fe\({}^{\text{II}}\)Fe\({}^{\text{II}}\) cofactor in HD-domain hydrolases suggests that the HD-domain sites may tune activity through the modulation of cofactor reduction potentials [40]. The iron ion in the Fe1 site serves as a Lewis acid and binds the substrate such that the C-X bond is opposite the iron (Figure 6) [15]. Then, the second iron site (Fe2) is available to bind molecular oxygen,  
Figure 6: Active sites of HD-domain oxygenases. Oxygenases utilize the “H... HD... H... H... D” motif to bind a diiron metal center.