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The article presents a study which examines the biosynthesis of Allocoronamic Acyl Thioster by an Fe''-dependent Halogenase and a Cyclopropane-Forming Flavoprotein. It notes that the four-protein cassette KtzABCD was used as flavoprotein to test if it can yield cyclopropyl-containing amino acid. Moreover, it mentions that the KtzABCD was cloned in Escherichia coli expression. It also concludes that the KtzABCD proteins synthesis rendered alloCMA as product of the enzymes.

Kutznerides 2 and 8 of the cyclic hexadepsipeptide family of antifungal natural products from the soil actinomycete Kutzneria sp. 744 contain two sets of chlorinated residues, a 6,7-dichlorohexahydropyrroloindole moiety derived from dichlorotryptophan and a 5-chloropiperazate moiety, as well as a methylcyclopropylglycine residue that may arise from isoleucine via a cryptic chlorination pathway. Previous studies identified KtzD, KtzQ, and KtzR as three halogenases in the kutzneride pathway but left no candidate for installing the C5 chlorine on piperazate. On the basis of analysis of the complete genome sequence of Kutzneria, we now identify a fourth halogenase in the pathway whose gene is separated from the defined kutzneride cluster by 12 open reading frames. KthP (kutzneride halogenase for piperazate) is a mononuclear nonheme iron halogenase that acts on the piperazyl ring tethered by a thioester linkage to the holo forms of thiolation domains. MS analysis of the protein-bound product confirmed chlorination of the piperazate framework from the (3S)- but not the (3R)-piperazyl-S-pantetheinyl thiolation proteins. After thioesterase-mediated release, nuclear magnetic resonance was used to assign the free imino acid as (3S,SS)-5-chloropiperazate, distinct from the 3S,SR stereoisomer reported in the mature kutznerides. These results demonstrate that a fourth halogenase, KthP, is active in the kutzneride biosynthetic pathway and suggest further processing of the (3S,SS)-5-chloropiperazate during subsequent incorporation into the kutzneride depsipeptide frameworks. [ABSTRACT FROM AUTHOR]