Today I met with Dr. Marvin Hackert, a specialist in protein crystallization at the University of Texas at Austin, to discuss my plans to isolate and crystallize an enzyme critical in the biosynthesis of morphine, which I imagine as a step on the way to developing a synthetic or semi-synthetic catalytic preparation for use in the manufacture of morphine. The enzyme, salutaradine synthase, has not previously been crystallized and the purpose of doing so would be to determine its 3D structure, particularly at and around the active site.
My notes from the meeting suggest a two-sided approach to the problem, which might be called top-down and bottom-up. The top-down approach is theoretical; it begins with the enzyme's gene sequence, or that of an isoform, and would approximate the 3D structure by computerized "fitting" of the primary sequence derived from the genome to the known structure of an analogous protein, if one can be found. Although inexact, this approach has the virtue of being inexpensive. It could give useful insight into the structure of the active site and, hence, to the mechanism of catalysis, thus paving the way for the development of an entirely synthetic catalytic system. The program to perform the "morphing" operation in which the sequence is extrapolated to a structure by analogy to a known protein is called SWISS-PDB, and is freely available through the internet.
On the practical side, the approach would be to isolate and purify the enzyme from a homogenous biological sample, crystallize it, and attempt to regenerate its catalytic activity in vitro. It would appear that the 1995 Amann, et. al, paper includes an assay that depends on the catalytic activity of the enzyme to track it through the isolation and purification process. This is something of a revelation, as my previous understanding was that the enzyme was inert apart from its associated cell membrane and that it had not been regenerated in vitro. It's a good sign because it indicates that such regeneration is possible and, moreover, routine enough to be used as an assay. Even if I fail ultimately to determine empirically the protein's structure, development of a reusable catalyst derived from the biological matrix could provide publishable and patentable results. Although this secondary goal does not require elucidation of the enzyme's structure, the effort to crystallize the protein is wasted in the absence of sequence data, because ultimate structural determination depends on both an x-ray diffraction pattern and knowledge of the primary structure.
1. What parts of the poppy genome have been sequenced?
2. Is there compelling evidence of the existence of salutaradine synthases in man?
3. Are the seminal investigators still working on this problem and are they willing to talk?
4. How much protein should I reasonably expect to need for the crystallization project?