Premium
Enzymology of Columbamide Biosynthesis
Author(s) -
D'Agostino Gabriel,
Manley Olivia M.,
Makris Thomas M.
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.633.7
Subject(s) - biosynthesis , halogenation , enzyme , acyl carrier protein , substrate (aquarium) , biochemistry , chemistry , stereochemistry , biology , combinatorial chemistry , organic chemistry , ecology
Marine cyanobacteria represent a unique, underexplored reservoir of bioactive natural products. Recent genomic analysis and mass spectrometry studies have revealed a novel chlorinated compound known as columbamide from Moorea bouillonii PNG isolated off the coast of New Guinea. Mass spectral shows columbamide to be a dihalogenated compound with chlorine atoms appended to the C6 and C12 positions of a hydrocarbon chain that is thought to be constructed from dodecanoic acid. Genomic studies have proposed a pathway for columbamide biosynthesis but it has yet to be supported by in vitro studies. Of particular interest, the enzymes proposed to catalyze the halogenation reactions, ColD and ColE, are most homologous to dinuclear iron‐dependent enzymes. Although diiron enzymes have been shown to have a great deal of functional diversity, halogenation is not amongst these reported activities. Our study seeks to characterize and reconstruct the early stages of the biosynthetic pathway by generating protein‐tethered substrates for the halogenases ColD and ColE. ColC is believed to be an acyl‐carrier protein for dodecanoic acid as well as a substrate of ColD/ColE. The first enzyme encoded by the gene cluster, ColA, is believed to attach dodecanoic acid to ColC. To investigate the biosynthetic pathway leading to halogenation, the role of ColA will be confirmed, and its enzymology characterized. Additionally, the well‐characterized enzyme acyl‐acyl carrier protein synthetase from the organism Vibrio harveyi will be used to efficiently generate large amounts of acylated‐ColC with non‐native fatty acids. Our study will seek to use the generated substrate‐library in concert with various spectroscopic techniques—such as optical spectra analysis, rapid‐mix stopped‐flow kinetics, and electron paramagnetic resonance (EPR)—to characterize and elucidate the mechanistic details of enzymes ColD/ColE. Support or Funding Information National Science Foundation; South Carolina Honors College‐‐Undergraduate Research Funding This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .