Adapting to oxygen: 3-Hydroxyanthrinilate 3,4-dioxygenase employs loop dynamics to accommodate two substrates with disparate polarities

Abstract

3-Hydroxyanthranilate 3,4-dioxygenase (HAO) is an iron-dependent protein that activates O and inserts both oxygen atoms into 3-hydroxyanthranilate (3-HAA). An intriguing question is how HAO can rapidly bind O, even though local O concentrations and diffusion rates are relatively low. Here, a close inspection of the HAO structures revealed that substrate- and inhibitor-bound structures exhibit a closed conformation with three hydrophobic loop regions moving toward the catalytic iron center, whereas the ligand-free structure is open. We hypothesized that these loop movements enhance O binding to the binary complex of HAO and 3-HAA. We found that the carboxyl end of 3-HAA triggers changes in two loop regions and that the third loop movement appears to be driven by an H-bond interaction between Asn and Ile Mutational analyses revealed that N27A, I142A, and I142P variants cannot form a closed conformation, and steady-state kinetic assays indicated that these variants have a substantially higher for O than WT HAO. This observation suggested enhanced hydrophobicity at the iron center resulting from the concerted loop movements after the binding of the primary substrate, which is hydrophilic. Given that O is nonpolar, the increased hydrophobicity at the iron center of the binary complex appears to be essential for rapid O binding and activation, explaining the reason for the 3-HAA-induced loop movements. Because substrate binding-induced open-to-closed conformational changes are common, the results reported here may help further our understanding of how oxygen is enriched in nonheme iron-dependent dioxygenases.

Observing 3-hydroxyanthranilate-3,4-dioxygenase in action through a crystalline lens

“…Our success in obtaining seven intermediate crystal structures provided an outstanding opportunity to address such a question in an iron-based dioxygenase. Previously, we have shown that the closed conformation due to 3-HAA binding increases the hydrophobicity of the iron center and causes a substantial increase of the O 2 affinity of the active site (16). The structural studies in this work show how the dioxygenase modulates the active-site conformation throughout the catalytic reaction.…”

“…The bond distance between Fe II and C3-hydroxyl group is 2.3 Å, while the C2-amino group is 3.2 Å from the Fe II ion (SI Appendix, Table S2). The previously observed loop movements that enhance the hydrophobicity of the Fe center and facilitate O 2 binding upon bidentate chelation of the substrate are not observed in this intermediate (16). Only one of three loops composed of residues 139 to 148 partially shift to the previously defined "closed" form (Fig.…”

“…Unlike other active-site residues presented in the structural figures, Thr39 is not strictly conserved in type III extradiol dioxygenases. Methionine is found in the same position in some HAO during sequences alignment (16). This variation may be derived from a need to tune the efficiency of the metal-and active site residue-assisted conversion of the product conformation.…”

“…The considerably slower reaction speed in crystals provides a promising platform to investigate reaction intermediates and the enzyme-bound product throughout the HAOmediated reaction. This set of experiments indicates that the catalytic rate constant is over 10,000-fold slower than that in solution (25 s −1 ) (12,16). Next, intermediates were probed for the presence of a radical state in pre-steady-state conditions (Fig.…”

“…Some bacterial HAOs contain a noncatalytic, rubredoxin-like site on the protein surface for iron acquisition and storage (15). In the solution state, HAO is an efficient catalyst with a reported turnover rate of 25 s −1 (12,16). The fast turnover rate is partially due to substrate-induced loop dynamics that help to increase O 2 concentration in the hydrophilic active site by making it more hydrophobic to accommodate the sequential binding of 3-HAA and O 2 , two substrates with disparate polarities, at the catalytic iron center (16).…”