| Autor: |
Mydy LS; Department of Structural Biology, University at Buffalo, The State University of New York, Buffalo, New York 14203, United States., Bailey DC; Department of Structural Biology, University at Buffalo, The State University of New York, Buffalo, New York 14203, United States., Patel KD; Department of Structural Biology, University at Buffalo, The State University of New York, Buffalo, New York 14203, United States., Rice MR; Department of Structural Biology, University at Buffalo, The State University of New York, Buffalo, New York 14203, United States., Gulick AM; Department of Structural Biology, University at Buffalo, The State University of New York, Buffalo, New York 14203, United States. |
| Abstrakt: |
Biosynthesis of the hydroxamate siderophore aerobactin requires the activity of four proteins encoded within the iuc operon. Recently, we biochemically reconstituted the biosynthetic pathway and structurally characterized IucA and IucC, two enzymes that sequentially couple N 6 -acetyl-N 6 -hydroxylysine to the primary carboxylates of citrate. IucA and IucC are members of a family of non-ribosomal peptide synthetase-independent siderophore (NIS) synthetases that are involved in the production of other siderophores, including desferrioxamine, achromobactin, and petrobactin. While structures of several members of this family were solved previously, there is limited mechanistic insight into the reaction catalyzed by NIS synthetases. Therefore, we performed a terreactant steady-state kinetic analysis and herein provide evidence for an ordered mechanism in which the chemistry is preceded by the formation of the quaternary complex. We further probed two regions of the active site with site-directed mutagenesis and identified several residues, including a conserved motif that is present on a dynamic loop, that are important for substrate binding and catalysis. |
