Kutznerides 2 and 8 of the cyclic hexadepsipeptide family of antifungal natural products from the soil actinomycete Kutzneria sp. 744 contain a 5-chloro-piperazate moiety. Our previous studies have identified three halogenases in the kutzneride pathway, but left no candidate for installing the C5 chlorine on piperazate. Based on an analysis of the complete genome sequence of Kutzneria, we now identify a fourth halogenase (KthP) whose gene is separated from the defined kutzneride cluster by twelve orfs. KthP is a mononuclear nonheme iron halogenase that acts on the piperazyl ring tethered to a thiolation domain. It works only on the 3S- but not the 3R-piperazyl moiety. After thioesterase-mediated release, NMR was used to assign the free imino acid as (3S, 5S)-5-chloropiperazate, distinct from the (3S, 5R)-stereoisomer reported in the mature kutznerides. These results demonstrate that a fourth halogenase is active in the kutzneride biosynthetic pathway, and suggest further processing of (3S,5S)-5-chloropiperazate during subsequent incorporations.

Biosynthetic chlorination of the piperazate residue in kutzneride biosynthesis by KthP

242nd National Meeting (2011)

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