Active site mapping

Du G., Ng C. S. and Prestwich G. D. (1994) Odorant binding by a pheromone binding protein active site mapping by photoaffinity labeling. Biochemistry 33, 4812-4819. 

Hydroxylation of alif tic carbon has rarely been accomplished in high yield in alkaloids, but some attempts have been made to produce an active site map for the hydroxylase of Streptomyces roseocHro-mogenes which may be of predictive value. ... 

In the following sections, it is explained briefly what makes NRPS biochemistry accessible to mass spectro-metric investigation and a new active site mapping approach for NRPS and PKS systems, the online LC—MS-PEA assay. Subsequently, each of the above categories of MS applications in NRPS biosynthesis research is presented based on recent research. 

Figure 12 Active site mapping by online LC-FT-ICR-MS-PEA assay.

The applied MS instrumentation for a substrate specificity assay depends on the size of the carrier domain construct whose active site is monitored. If multidomain constructs or T domain constructs >20 kDa are investigated, active site mapping is required and, therefore, ESI-FTMS instruments have to be applied because of their established active site mapping capabilities described above. If the monitored T domain constructs are freestanding and <20 kDa, no active site mapping is required and low-resolution MS instruments can be utilized for characterization of loaded substrates too. 

Although much of the work on the microbial hydroxylation of amides has been directed at active-site mapping of the enzyme responsible, the products themselves are valuable building blocks for further synthesis, for example, for various optically active sesquiterpenes or -lactams. In this latter context regioselective hydroxylation of unactivated positions is particularly attractive as several -lactam antibiotics, e.g. the carbapenem derivative thienamycin, have a free hydroxy group in their structure. 

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