Characterization of a dual specificity aryl acid adenylation enzyme with dual function in nikkomycin biosynthesis.
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.
Moon
Mary
M
Department of Pharmaceutical Sciences, University of Kentucky, Lexington, KY 40536, USA.
Van Lanen
Steven G
SG
D000617:Aminoglycosides / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D000935:Antifungal Agents / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000637:Transaminases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D000617:Aminoglycosides; D000935:Antifungal Agents; D001426:Bacterial Proteins; C011952:nikkomycin; D000637:Transaminases; D003066:Coenzyme A Ligases