Zhang, Jiancheng; (2001) Characterisation of bacterial NOS. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Nitric oxide (NO) is a pleiotropic regulator of many biological processes, and has been well characterised in eukaryotic systems where it is generated by a family of nitric oxide synthases (NOS). To date, there has been no good molecular evidence for the existence of similar proteins in bacteria. All NOS isoforms characterised so far comprise two distinct domains, an N-terminal heme domain, and a C-terminal reductase domain with sequence similarity to cytochrome P450 reductase. A protein with a high degree of similarity to the N-terminal domain of human inducible nitric oxide synthase (iNOS) has been identified in Staphylococcus aureus, and called SANOS. The gene encoding the protein was amplified using PCR, cloned, sequenced, and expressed. The purified protein was characterised using a range of biochemical techniques including heme-spectral analysis, enzyme kinetics, and crystallisation. SANOS is a heme protein, and like mammalian NOS N-terminal domains, possesses the ability to bind L-arginine, the NO intermediate N-hydroxy arginine (NOHA), and NOS inhibitors. Interestingly, SANOS can generate NO from NOHA in a reaction identical to that of the murine iNOS heme domain. The crystal structure of SANOS has been determined as part of a collaboration with Dr. Dave Stammers at Oxford University and shows SANOS to be a dimer. In all the NOS family members studied to date, tetrahydrobiopterin (BH4) occurs at the dimer interface. Bacteria do not possess BH4, and interestingly, the crystal structure of SANOS shows the presence of NAD at the dimer interface presumably carrying out the same structural role. Interestingly, a protein with a high degree of identity to the mammalian NOS C- terminal reductase domain (NADPH cytochrome P450 reductase) was also identified in S.aureus. PCR was used to amplify, clone, and express this protein, which was named STAPHRED. Recombinant protein has been generated for enzyme assays in studies with SANOS. To date, all NOS family members appear as fused heme and reductase domains. This study shows that in S. aureus these two domains can be found as separate proteins. This is the first report on the molecular characteristics of NOS-like proteins in bacteria, and illustrates the power of the process from BLAST predictions to the analysis of functional protein.

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