Characterization of the 2-hydroxy-acid dehydrogenase McyI, encoded within the microcystin biosynthesis gene cluster of Microcystis aeruginosa PCC7806.
The cyanobacterium Microcystis aeruginosa is widely known for its production of the potent hepatotoxin microcystin. This cyclic heptapeptide is synthesized non-ribosomally by the thio-template function of a large modular enzyme complex encoded within the 55-kb microcystin synthetase gene (mcy) cluster. The mcy gene cluster also encodes several stand-alone enzymes, putatively involved in the tailoring and export of microcystin. This study describes the characterization of the 2-hydroxy-acid dehydrogenase McyI, putatively involved in the production of d-methyl aspartate at position 3 within the microcystin cyclic structure. A combination of bioinformatics, molecular, and biochemical techniques was used to elucidate the structure, function, regulation, and evolution of this unique enzyme. The recombinant McyI enzyme was overexpressed in Escherichia coli and enzymatically characterized. The hypothesized native activity of McyI, the interconversion of 3-methyl malate to 3-methyl oxalacetate, was demonstrated using an in vitro spectrophotometric assay. The enzyme was also able to reduce alpha-ketoglutarate to 2-hydroxyglutarate and to catalyze the interconversion of malate and oxalacetate. Although NADP(H) was the preferred cofactor of the McyI-catalyzed reactions, NAD(H) could also be utilized, although rates of catalysis were significantly lower. The combined results of this study suggest that hepatotoxic cyanobacteria such as M. aeruginosa PCC7806 are capable of producing methyl aspartate via a novel glutamate mutase-independent pathway, in which McyI plays a pivotal role.
Pearson
Leanne A
LA
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia.
Barrow
Kevin D
KD
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia.
Neilan
Brett A
BA
School of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, New South Wales 2052, Australia. Electronic address: b.neilan@unsw.edu.au.
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D019143:Evolution, Molecular; D015870:Gene Expression; D052998:Microcystins / Q000096:biosynthesis / Q000235:genetics*; D046931:Microcystis / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family / Q000235:genetics*; D046908:Peptide Biosynthesis, Nucleic Acid-Independent / Q000235:genetics*; D010802:Phylogeny
D001426:Bacterial Proteins; D052998:Microcystins; C078588:microcystin; D000429:Alcohol Oxidoreductases; C083779:2-hydroxyacid dehydrogenase