16597-58-3Relevant articles and documents
The power of two arginine 51 and arginine 239* from a neighboring subunit are essential for catalysis in α-amino-β- carboxymuconate-∈-semialdehyde decarboxylase
Huo, Lu,Davis, Ian,Chen, Lirong,Liu, Aimin
, p. 30862 - 30871 (2013)
Although the crystal structure of α-amino-β-carboxymuconate- ∈-semialdehyde decarboxylase from Pseudomonas fluorescens was solved as a dimer, this enzyme is a mixture of monomer, dimer, and higher order structures in solution. In this work, we found that the dimeric state, not the monomeric state, is the functionally active form. Two conserved arginine residues are present in the active site: Arg-51 and an intruding Arg-239* from the neighboring subunit. In this study, they were each mutated to alanine and lysine, and all four mutants were catalytically inactive. The mutants were also incapable of accommodating pyridine-2,6-dicarboxylic acid, a competitive inhibitor of the native enzyme, suggesting that the two Arg residues are involved in substrate binding. It was also observed that the decarboxylase activity was partially recovered in a heterodimer hybridization experiment when inactive R51(A/K) and R239(A/K) mutants were mixed together. Of the 20 crystal structures obtained from mixing inactive R51A and R239A homodimers that diffracted to a resolution lower than 3.00 A, two structures are clearly R51A/R239A heterodimers and belong to the C2 space group. They were refined to 1.80 and 2.00 A resolutions, respectively. Four of the remaining crystals are apparently single mutants and belong to the P42212 space group. In the heterodimer structures, one active site is shown to contain dual mutation of Ala-51 and Ala-239*, whereas the other contains the native Arg-51 and Arg-239* residues, identical to the wildtype structure. Thus, these observations provide the foundation for a molecular mechanism by which the oligomerization state of α-amino-β- carboxymuconate-∈-semialdehyde decarboxylase could regulate the enzyme activity.
Adapting to oxygen: 3-Hydroxyanthrinilate 3,4-dioxygenase employs loop dynamics to accommodate two substrates with disparate polarities
Yang, Yu,Liu, Fange,Liu, Aimin
, p. 10415 - 10424 (2018)
3-Hydroxyanthranilate 3,4-dioxygenase (HAO) is an iron-dependent protein that activates O2 and inserts both oxygen atoms into 3-hydroxyanthranilate (3-HAA). An intriguing question is how HAO can rapidly bind O2, even though local O2 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 O2 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 Asn27 and Ile142. 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 Km for O2 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 O2 is nonpolar, the increased hydrophobicity at the iron center of the binary complex appears to be essential for rapid O2 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.
