UC Berkeley

Mycobacterium tuberculosis is the leading causative agent of tuberculosis, from

which millions die annually. A putative lipopeptide biosynthetic gene cluster has been

shown to be essential for the survival of this pathogen in hosts, and homologous gene

clusters have also been found in all pathogenic mycobacteria and other species of

Actinobacteria. However, the specific functions of the encoded proteins have been

elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses,

we have revealed that the five encoded biosynthetic enzymes are capable of synthesizing

a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The

biosynthesis features the generation of isonitrile from a single precursor Gly promoted by

a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of

both amino groups of Lys by the same isonitrile acyl chain facilitated by a single

condensation domain of a nonribosomal peptide synthetase. Additionally, the

biosynthesis of this bioactive isonitrile moiety is extraordinary and is distinct from the

IsnA family of isonitrile synthases. We herein provide the first structural and biochemical

evidence of an alternative mechanism for isonitrile formation.