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Insights into the Reaction Mechanism of the Prolyl–Acyl Carrier Protein Oxidase Involved in Anatoxin-a and Homoanatoxin-a Biosynthesis
journal contribution
posted on 2011-08-23, 00:00 authored by Stéphane Mann, Bérangère Lombard, Damarys Loew, Annick Méjean, Olivier PlouxAnatoxin-a and homoanatoxin-a are two potent cyanobacterial
neurotoxins.
We recently reported the identification of the gene cluster responsible
for the biosynthesis of these toxins as well as the in-vitro reconstitution
of the first steps of this biosynthesis. We now report experimental
evidence supporting the proposed reaction mechanism of AnaB, a flavoprotein
homologous to acyl-CoA dehydrogenase. AnaB catalyzes the two-electron
oxidation of prolyl-AnaD, which is proline linked to the acyl carrier
protein holo-AnaD, to dehydroprolyl-AnaD using oxygen as the second
substrate. AnaB is thus an oxidase. By using liquid chromatography
coupled to tandem mass spectrometry (LC-MS/MS), we have identified
and characterized dehydroprolyl-AnaD, the AnaB product. We estimated
an apparent catalytic constant of 1 min–1 for AnaB
catalysis. We synthesized several deuterium-labeled prolines and enzymatically
transformed them into their corresponding prolyl-AnaD. These deuterium-labeled
prolyl-AnaDs were oxidized in the presence of AnaB, and the deuterium
labeling in the remaining substrate and in the product was determined
by LC-MS/MS. The data supported a reaction mechanism starting with
a rapid enolization followed by a slow oxidation to give the conjugated
imine, which in turn was isomerized to pyrroline-5-carboxyl-AnaD.
We also showed that cis- and trans-4-fluoro-l-prolyl-AnaD and 3,4-dehydro-l-prolyl-AnaD
were transformed into pyrrole-2-carboxyl-AnaD by AnaB. Thus, the 4-fluoro-analogues
experienced a β-elimination supporting the AnaB-catalyzed aza–allylic
isomerization. We identified by sequence alignment the AnaB active
site base, Glu244. We produced, purified, and characterized the E244A
AnaB mutant, which is inactive, supporting the catalytic role of E244
as a base.