Identification of nitric oxide synthase in Flammulina velutipes

Abstract

Nitric oxide synthase (NOS) was discovered in the edible mushroom, Flammulina velutipes. NOS activity gradually increased after exposure to light and lowering temperature to 10 C at day 16. The maximal activity was seen at day 23 of incubation and followed by fruit body formation at day 30. NOS at day 23 was 72–fold higher than the initial level. A similar pattern was observed when nitrite level was measured. Treatment with the NO generator, sodium nitroprusside, stimulated the formation of fruit bodies in F. velutipes, whereas NOS inhibitors, N-nitro-l-arginine methylester and aminoguanidine delayed them, indicating that NOS activity is related to development of F. velutipes. The fungal NOS was purified by DEAE-Sephadex A-50, 2′,5′-ADP agarose affinity, and mono P chromatofocusing column chromatographies. The enzyme required NADPH, FAD, FMN, O₂, and tetrahydrobiopteridine as cofactors in generation of nitric oxide, but no Ca²⁺ and calmodulin. The K_m for l-arginine was determined to be 3.3 × 10^-5 M and the V_max was 2.4 × 10^-4 μmol/min/mg. NOS was inhibited by with K_i values of 2.9 × 10^-4 and 2.8 × 10^-5 M, respectively.

Key Words:

• differentiation
• fruit body