Function and Structure of MalA/MalA′, Iterative Halogenases for Late-Stage C–H Functionalization of Indole Alkaloids

Abstract

Malbrancheamide is a dichlorinated fungal indole alkaloid isolated from both Malbranchea aurantiaca and Malbranchea graminicola that belongs to a family of natural products containing a characteristic bicyclo[2.2.2]diazaoctane core. The introduction of chlorine atoms on the indole ring of malbrancheamide differentiates it from other members of this family and contributes significantly to its biological activity. In this study, we characterized the two flavin-dependent halogenases involved in the late-stage halogenation of malbrancheamide in two different fungal strains. MalA and MalA’ catalyze the iterative dichlorination and monobromination of the free substrate premalbrancheamide as the final steps in the malbrancheamide biosynthetic pathway. Two unnatural bromo-chloro-malbrancheamide analogs were generated through MalA-mediated chemoenzymatic synthesis. Structural analysis and computational studies of MalA’ in complex with three substrates revealed that the enzyme represents a new class of zinc-binding flavin-dependent halogenases, and provides new insights into a potentially unique reaction mechanism.

Structural and stereochemical diversity in prenylated indole alkaloids containing the bicyclo[2.2.2]diazaoctane ring system from marine and terrestrial fungi

“…It should be noted that many laboratories in the synthetic community have published elegant, yet non-biomimetic total syntheses of numerous natural products in this family of alkaloids. 41,46,47,57,58,66,67,85,86,88,90,95,97,99,102,108,115,131–146 These efforts have been reviewed by Miller and Williams in 2009 (ref. 40) and additional total syntheses have appeared in the literature since 2009.…”

“…Again here, the biomimetic synthesis of pre-malbrancheamide to prepare doubly 13 C-labeled isotopomers of 128 and premalbrancheamide was adopted. 115 When both substrates were presented to cultures of Malbranchea aurantiaca , only premalbrancheamide incorporated into malbrancheamide B (5.5% incorporation). The lack of incorporation of either dioxopiperazine species 126 and 128 begged the question as to when and how the tryptophan-derived carbonyl group is reduced by a net four-electrons?…”

Halogenase engineering and its utility in medicinal chemistry

“…1c). 10 Chloramphenicol is a well-known antibiotic that is chlorinated by the FDH CmlS, which bears a rare covalently bound flavin cofactor, and its biosynthetic halogenation remains under investigation. 25–28 Molecular dissection has enabled engineering strategies directed toward improving stability, expanding substrate scope, and altering site-selectivity of FDHs.…”

“…2). 10 Detailed mechanistic and structural analyses of halogenases have been instrumental in the development of these enzymes as biocatalysts. With insight into how these proteins work, engineering efforts have produced selective halogenases that perform chemical transformations on therapeutically relevant small molecules.…”

“…Recent studies have included the first application of computational chemistry to analyze the mechanism of FDH-catalyzed chlorination. 10 …”