Cloning And Biochemical Analysis Of A Nitric Oxide Synthase-like Protein From Streptomyces Avermitis

Nitric oxide synthases (NOSs) were heme-based mono-oxygenases that oxidize L-arginine to nitric oxide (NO), a signaling molecule and cytotoxic agent in higher organisms. Although NOS-like activity had been reported in many bacteria, only a few bacterial homologs of mammalian NOSs (mNOSs) had been characterized to date. The research showed, in contrast to mNOSs, which possessed both a catalytic and a reductase domain, the bacterial enzymes lacked reductase domains and require the supply of suitable reductants to produce NO. Thus bacterial NOSs seemed to have functions that differed from those of mNOSs, including nitration of different metabolites and protection against oxidative stress. Studies of bacterial NOSs would probably result in a better understanding of the mechanism of NO synthesis and unveiled a variety of new functions for NO in microbes. There was also a similar bNOS gene existing in Streplomyce avermitilis, and no paper of it had been reported, therefore we decided to study further about it to know its biochemical function in Streplomyces.In the paper, we extracted genomic DNA of Streptomyce avermililis and obtained a similar nitric oxide synthase gene from Slreplomyce avermititis by a series of molecular cloning methods. The similar nitric oxide synthase gene was named as sanos. After bioinformatics analysis of it, we found that sanos was the protential nos of Streplomyces avermitilis MA-4680DNA (number NC-003155) in NCBI, which had a high homology with that of Streplomyces scabiei and Homo sapiens. So it could be initially identified as nitric oxide synthase gene. In addition, sanos had the composition of516aa and molecular weight of56.4kDa. The predicted value showed that saNOS was an unstable hydrophilic protein. And compared to most of the bacterial nitric oxide synthases, it had a N-terminal hook and zinc pterin binding, which showed closer relationship with mammalian nitric oxide synthases.To further verify saNOS has ability of oxidizing L-arginine to nitric oxide (NO), we designed to construct pET28a-saNOS expression vector and express saNOS in Escherichia coli by IPTG. The results showed that when the IPTG concentration was0.2mM and the expressing time was8h, saNOS had the largest amount of protein expression. We measured the activity of saNOS by Nitric Oxide Synthase Assay Kit. The data told us saNOS enzyme from Streptomyces avermitilis do indeed produce NO in living cells and accomplish this task by hijacking available cellular redox partners that are not normally committed to NO production.Meanwhile, we obtained a muntant strain Streptomyces coelicolor, which could express saNOS. The expression of saNOS in Streptomyces coelicolor caused a delay switch from vegetative mycelia to aerial mycelia and higher expression of Act and lower expression of Red than that in the wild type. The Streptomyces avermitilis growth and melanin production were investigated in the presence of a NOS inhibitor (L-NMMA), a NO scavenger (cPTIO) and a NO donor (SNP). cPTIO and NMMA had no significant effect on melanin production and bacterial growth when the quantity of NO had decreased apparently. But SNP obviously increased melanin production but delayed bacterial morphological transition while the quantity of NO was almost two times higher than that of without SNP addition. Our results suggested that saNOS might have a negative role in morphological transition and an influence on the secondary metabolites synthesis in Streptomyces through oxidizing L-arginine to nitric oxide (NO).