The highly selective oxidative halogenations by non-heme iron and α-ketoglutarate-dependent enzymes are key reactions in the biosynthesis of lipopeptides, and often bestow valuable bioactivity to the metabolites. Here we present the first biochemical characterization of a putative fatty acyl halogenase, HctB, which is found in the hectochlorin biosynthetic pathway of Lyngbya majuscula. Its unprecedented three-domain structure, which includes an acyl carrier protein domain, allows self-contained conversion of the covalently tethered hexanoyl substrate. Structural analysis of the native product by (13) C NMR reveals high regioselectivity but considerable catalytic promiscuity. This challenges the classification of HctB as a primary halogenase: along with the proposed dichlorination, HctB performs oxygenation and an unprecedented introduction of a vinyl-chloride moiety into the nonactivated carbon chain. The relaxed substrate specificity is discussed with reference to a molecular model of the enzyme-substrate complex. The results suggest that fatty acyl transformation at the metal center of HctB can bring about considerable structural diversity in the biosynthesis of lipopeptides.
Mononuclear nonheme
Fe(II) (MNH) and α-ketoglutarate (α-KG)
dependent halogenases activate O2 to perform oxidative
halogenations of activated and nonactivated carbon centers. While
the mechanism of halide incorporation into a substrate has been investigated,
the mechanism by which halogenases prevent oxidations in the absence
of chloride is still obscure. Here, we characterize the impact of
chloride on the metal center coordination and reactivity of the fatty
acyl-halogenase HctB. Stopped-flow kinetic studies show that the oxidative
transformation of the Fe(II)-α-KG-enzyme complex is >200-fold
accelerated by saturating concentrations of chloride in both the absence
and presence of a covalently bound substrate. By contrast, the presence
of substrate, which generally brings about O2 activation
at enzymatic MNH centers, only has an ∼10-fold effect in the
absence of chloride. Circular dichroism (CD) and magnetic CD (MCD)
studies demonstrate that chloride binding triggers changes in the
metal center ligation: chloride binding induces the proper binding
of the substrate as shown by variable-temperature, variable-field
(VTVH) MCD studies of non-α-KG-containing forms and the conversion
from six-coordinate (6C) to 5C/6C mixtures when α-KG is bound.
In the presence of substrate, a site with square pyramidal five-coordinate
(5C) geometry is observed, which is required for O2 activation
at enzymatic MNH centers. In the absence of substrate an unusual trigonal
bipyramidal site is formed, which accounts for the observed slow,
uncoupled reactivity. Molecular dynamics simulations suggest that
the binding of chloride to the metal center of HctB leads to a conformational
change in the enzyme that makes the active site more accessible to
the substrate and thus facilitates the formation of the catalytically
competent enzyme–substrate complex. Results are discussed in
relation to other MNH dependent halogenases.
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