Two-Enzyme Pathway Links l-Arginine to Nitric Oxide in N-Nitroso Biosynthesis.

Nitric oxide (NO) has wide-ranging roles in biology, but less is known about its role in building chemical diversity. Here we report a new route to NO from the biosynthetic pathway to the N-nitroso compound streptozocin. We show that the N-nitroso group of streptozocin comes from the biosynthetic reassembly of l-arginine, with the guanidino nitrogens forming a nitrogen-nitrogen bond. To understand this biosynthetic process, we identify the biosynthetic gene cluster of streptozocin and demonstrate that free l-arginine is N-methylated by StzE to give Nω-monomethyl-l-arginine. We show that this product is then oxidized by StzF, a nonheme iron-dependent enzyme unrelated to known nitric oxide synthases, generating a urea compound and NO. Our work implies that formation and capture of NO is the likely route to N-nitroso formation in vivo. Altogether, our work unveils a new enzyme pair for the production of NO from l-arginine and sets the stage for understanding biosynthetic routes to N-nitroso natural products.

Nutrient supplements boost yeast transformation efficiency.

Efficiency of yeast transformation is determined by the rate of yeast endocytosis. The aim of this study was to investigate the effect of introducing amino acids and other nutrients (inositol, adenine, or p-aminobenzoic acid) in the transformation medium to develop a highly efficient yeast transformation protocol. The target of rapamycin complex 1 (TORC1) kinase signalling complex influences the rate of yeast endocytosis. TORC signaling is induced by amino acids in the media. Here, we found that increasing the concentration of amino acids and other nutrients in the growth media lead to an increase yeast transformation efficiency up to 107 CFU per µg plasmid DNA and per 108 cells with a 13.8 kb plasmid DNA. This is over 130 times that of current published methods. This improvement may facilitate more efficient experimentation in which transformation efficiency is critical, such as yeast two-hybrid screening.