Biosynthesis of 4-methylproline in cyanobacteria: cloning of nosE and nosF genes and biochemical characterization of the encoded dehydrogenase and reductase activities

The biosynthesis of the unusual amino acid 4-methylproline in the Nostoc genus of cyanobacteria was investigated on the genetic and enzymatic level. Two genes involved in the biosynthesis were cloned and the corresponding enzymes, a zinc-dependent long-chain dehydrogenase and a Delta(1)-pyrroline-5-carboxylic acid (P5C) reductase homologue, were overexpressed in Escherichia coli and biochemically characterized. Putative substrates were synthesized to test enzyme substrate specificities, and deuterium labeling studies were carried out to reveal the stereospecificities of the enzymatic reactions with respect to the substrates as well as to the coenzymes.