Abstract
The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of
Klebsiella
ATCC 8724 present an unusual case in which two enzymes with different structures and distinct activities towards their common substrates (1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene and dioxygen) are derived from the same polypeptide chain. Structural and functional differences between the two isozymes are determined by the type of M
+2
metal ion bound in the active site. The Ni
+2
-bound NiARD catalyzes an off-pathway shunt from the methionine salvage pathway leading to the production of formate, methylthiopropionate and carbon monoxide, while the Fe
+2
-bound FeARD' catalyzes the on-pathway formation of methionine precursor 2-keto-4-methylthiobutyrate and formate. Four potential protein-based metal ligands were identified by sequence homology and structural considerations. Based on the results of site-directed mutagenesis experiments, X-ray absorption spectroscopy (XAS) and isothermal calorimetry measurements, it is concluded that the same four residues, His 96, His 98, Glu 102 and His 140, provide the protein-based ligands for the metal in both the Ni- and Fe-containing forms of the enzyme, and subtle differences in the local backbone conformations trigger the observed structural and functional differences between the FeARD' and NiARD isozymes. Furthermore, both forms of the enzyme bind their respective metals with pseudo-octahedral geometry, and both may lose a His ligand upon binding of substrate under anaerobic conditions. However, mutations at two conserved non-ligand acidic residues, Glu 95 and Glu 100, result in low metal contents for the mutant proteins as isolated, suggesting that some of the conserved charged residues may aid in transfer of metal from
in vivo
sources or prevent the loss of metal to stronger chelators. The Glu 100 mutant reconstitutes readily but has low activity. Mutation of Asp 101 results in an active enzyme that incorporates metal
in vivo
but shows evidence of mixed forms.