Characterization of a dual specificity aryl acid adenylation enzyme with dual function in nikkomycin biosynthesis.

Mary Moon, Steven G Van Lanen

Index: Biopolymers 93(9) , 791-801, (2010)

Full Text: HTML

Abstract

Nikkomycin Z is a dipeptide antifungal antibiotic characterized by two nonproteinogenic amino acids, nikkomycin C(Z) and 4-(4'-hydroxy-2'-pyridinyl)-homothreonine (HPHT). The HPHT scaffold is assembled by an aldol reaction between 2-oxobutyrate and picolinaldehyde, the latter of which is derived from picolinic acid that is activated and loaded to coenzyme A by the aryl-activating adenylation enzyme, NikE. We now provide evidence that NikE is also involved in the activation and loading of the alpha-keto acid precursor, 4-(2'-pyridinyl)-2-oxo-4-hydroxyisovalerate (POHIV), to a phosphopantetheinyl group of an acyl carrier protein domain of NikT. POHIV was synthesized using Escherichia coli 2-dehydro-3-deoxy-phosphogluconate aldolase, and phenylalanine dehydrogenase from Bacillus sp. NRRL B-14911 was used to prepare the alpha-amino acid, 4-(2'-pyridinyl)-homothreonine (PHT). Using the carboxylic acid-dependent, ATP-[(32)P]PP(i) exchange assay, NikE is shown to activate both picolinic acid and POHIV but not PHT. Furthermore, NikE loads POHIV to holo-NikT to generate a new thioester-linked intermediate, which was not observed using a NikT(S33A) mutant. Thus, NikE activates two distinct carboxylic acids to form two new thioester intermediates, one of which is subsequently reduced to the aldehyde and the other that likely serves as a substrate for the aminotransferase domain of NikT prior to condensation with nikkomycin C(Z) to yield the dipeptide.Copyright 2010 Wiley Periodicals, Inc.