Characterization of a dual specificity aryl acid adenylation enzyme with dual function in nikkomycin biosynthesis
✍ Scribed by Mary Moon; Steven G. Van Lanen
- Book ID
- 102762170
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 2010
- Tongue
- English
- Weight
- 681 KB
- Volume
- 93
- Category
- Article
- ISSN
- 0006-3525
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✦ Synopsis
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 α‐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 α‐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. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 791–801, 2010.