Biosynthesis of (-)-(1S,2R)-allocoronamic acyl thioester by an Fe(II)-dependent halogenase and a cyclopropane-forming flavoprotein.
The biosynthetic gene cluster for the kutzneride family of hexapeptidolactones includes the four-gene cassette ktzABCD postulated to generate a nonproteinogenic amino acid. Encoded by this cassette are the nonheme FeII-dependent halogenase KtzD and the acyl-CoA dehydrogenase-like flavoprotein KtzA, proposed to work in conjunction with adenylating protein KtzB and carrier protein KtzC. In the present work, we report the in vitro reconstitution of this four-protein system and identify the final product as (1S,2R)-allocoronamic acid bound in thioester linkage to KtzC. Further analysis of KtzD and KtzA support a biosynthetic pathway that involves KtzD-mediated generation of a gamma-chloroisoleucyl intermediate which is cyclized to the final product by KtzA without redox participation of the bound flavin cofactor. This work introduces a new monomer for potential incorporation into nonribosomal peptides and validates the unique strategy for its biosynthesis.
Neumann
Christopher S
CS
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Walsh
Christopher T
CT
D039903:Actinobacteria / Q000201:enzymology / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D002712:Chlorides / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003500:Cyclization; D003521:Cyclopropanes / Q000737:chemistry / Q000378:metabolism*; D003903:Deuterium / Q000737:chemistry; D005420:Flavoproteins / Q000737:chemistry / Q000378:metabolism*; D054879:Halogenation; D007211:Indoles / Q000737:chemistry; D007501:Iron / Q000737:chemistry / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods; D008968:Molecular Conformation; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D013237:Stereoisomerism; D013997:Time Factors
C533762:(1S,2R)-allocoronamic acid; D001426:Bacterial Proteins; D002352:Carrier Proteins; D002712:Chlorides; D003521:Cyclopropanes; D005420:Flavoproteins; D007211:Indoles; D009097:Multienzyme Complexes; D003903:Deuterium; D007501:Iron; D010088:Oxidoreductases