Structural basis of a 2-oxoglutarate-dependent heme-oxygenase-like enzyme HamA in fragin biosynthesis

Abstract

The fungicide fragin, which contains a diazeniumdiolate moiety, exhibits a broad spectrum of biological activities. HamA, the key enzyme responsible for forming the nitrogen–nitrogen bond in this moiety, was investigated in this study. We determined the crystal structure of HamA at 2.0 Å resolution, revealing a mononuclear iron center in the active site coordinated by both the “2His–1Glu” motif and an acetate group. Notably, HamA adopts a heme oxygenase-like fold, forming a hydrophobic cavity within a helical bundle that likely accommodates the substrate. Structural data confirmed the presence of an acetate and a formate group near the active site and microscale thermophoresis (MST) experiments further demonstrated HamA's ability to bind 2-oxoglutarate (2OG) with a dissociation constant (K_d) of 208 ± 1.42 μM. In summary, this study elucidates the 2OG-dependent heme-oxygenase-like enzyme HamA with a monoiron active center, providing critical structural insights into the mechanistic formation of the diazeniumdiolate moiety in fragin.