Biosynthesis of a Metallophore in Actinobacteria | AIChE

Biosynthesis of a Metallophore in Actinobacteria

An unprecedented mechanism for isonitrile biosynthesis by a dioxygenase which uses one substrate as both the nitrogen and carbon source has been discovered in the biosynthetic pathway of a metallophore in Actinobacteria. Characterization of the enzyme and natural product in Streptomyces coeruleorubidus and Mycobacterium marinum is crucial to understanding this mechanism. This biosynthetic pathway consists of five genes, four of which were reconstituted in vitro. Currently, the dioxygenase is unable to be reconstituted because it requires that the substrate is bound to an acyl carrier protein (ACP). The thioesterase in this pathway cleaves the bond between the substrate and the ACP as well as its primary function of a glycine addition to a fatty acyl ACP, inhibiting the function of the dioxygenase. To avoid premature hydrolysis by the thioesterase, the substrate was produced through a seven-step synthesis involving a thioesterase bond linking 3-aminobutanoic acid to coenzyme A and the attachment to an apo ACP using a phosphopantetheinyl transferase. Through heterologous expression in Escherichia coli, the product of this pathway was characterized; however, the product of the native host undergoes modifications not observed in E. coli. Comparing the metabolome of wild type M. marinum and ΔmmaA-E, a unique mass was revealed (1090 m/z). The culture was then supplemented with 15N glycine and the resultant mass shift was observed (1095 m/z), indicating that this product is likely similar to the product observed in E. coli. The mmaA-E product was previously shown to be involved with metal transport. To determine the potential role of neighboring proteins in metal acquisition, four M. marinum genes; the dioxygenase, a major facilitator superfamily protein, an ATPase, and a PPE family protein, were knocked-out and complemented. Inductively coupled plasma optical emission spectroscopy will be used to detect changes in the intracellular metal concentration associated with each genotype.


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