Structure-Guided Functional Characterization of Enediyne Self-Sacrifice Resistance Proteins, CalU16 and CalU19

Abstract

Calicheamicin γ1I (1) is an enediyne antitumor compound produced by Micromonospora echinospora spp. calichensis, and its biosynthetic gene cluster has been previously reported. Despite extensive analysis and biochemical study, several genes in the biosynthetic gene cluster of 1 remain functionally unassigned. Using a structural genomics approach and biochemical characterization, two proteins encoded by genes from the 1 biosynthetic gene cluster assigned as “unknowns”, CalU16 and CalU19, were characterized. Structure analysis revealed that they possess the STeroidogenic Acute Regulatory protein related lipid Transfer (START) domain known mainly to bind and transport lipids and previously identified as the structural signature of the enediyne self-resistance protein CalC. Subsequent study revealed calU16 and calU19 to confer resistance to 1, and reminiscent of the prototype CalC, both CalU16 and CalU19 were cleaved by 1in vitro. Through site-directed mutagenesis and mass spectrometry, we identified the site of cleavage in each protein and characterized their function in conferring resistance against 1. This report emphasizes the importance of structural genomics as a powerful tool for the functional annotation of unknown proteins.

Genome neighborhood network reveals insights into enediyne biosynthesis and facilitates prediction and prioritization for discovery

“…A small sample set of two, both from Micromonospora spp., makes it extremely difficult to identify trends associated with 10-membered enediyne chemistry. For example, CalU16 and CalU19 were recently reported as self-sacrifice resistance proteins for the CALs [12]. No homologous proteins are found in the DYN cluster suggesting that the CalU16/U19 family of resistance proteins is not conserved in all 10-membered enediynes and therefore cannot be used as a 10-membered indicator (Fig.…”

A community resource of experimental data for NMR / X‐ray crystal structure pairs

“…Some of these proteins form higher‐order oligomers in the crystal structure. A few of these structures have been published as independent papers, while others are currently being used for follow‐up structure–function studies.…”

A comprehensive review of glycosylated bacterial natural products

“…478 As an additional signature, 9-membered enediynes are commonly associated with a stabilizing apoprotein and 10-membered enediynes have been more recently been associdated with a novel self-sacrifice resistance mechansism. 479–481 Enediynes bind the minor groove of dsDNA in a sequence/context-specific fashion which is often mediated via their appended carbohydrates. Subsequent reductive/nucleophilic activation initiates a Bergman or Myers-Saito cyclization, the reactive diradical intermediate of which abstracts hydrogen from the DNA backbone to ultimately afford oxidative DNA strand cleavage.…”