Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.739930
Title: Structural analysis of a Papaver somniferum O-methyltransferase involved in noscapine biosynthesis
Author: Cabry, Marc P.
ISNI:       0000 0004 7231 5263
Awarding Body: University of York
Current Institution: University of York
Date of Award: 2017
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Abstract:
Noscapine is a major alkaloid that is found, along with the more commonly known drugs morphine and codeine, in the latex of opium poppy (Papaver somniferum). Noscapine has classically been used as a cough suppressant, but also has emerging antitumor properties. Unlike morphine and codeine, noscapine has no addictive properties. Recently, a 10-gene cluster for the synthesis of noscapine was discovered. The first distinct step in this pathway is an O-methylation of scoulerine to tetrahydrocolumbamine by a class I S-adenosylmethionine (SAM) dependent methyltransferase, referred to as Papaver somniferum O-methyltransferase 1 (PSMT1). PSMT1 is therefore an interesting target for detailed structural and functional analysis. PSMT1 has been expressed as a fusion protein, tagged to improve solubility, and purified for X-ray diffraction studies. Here we present the structures of an "apo" enzyme and an enzyme complex with SAM, bound in "open" enzyme conformations. A surface entropy reduction mutant enzyme allowed access to a closed conformation high-resolution structure in complex with S-adenosylhomocysteine (SAH) and the product tetrahydrocolumbamine. These X-ray structures provide deep insight into the structure function relationships of PSMT1. PSMT1 is a homodimer with distinct dimerisation, SAM binding and substrate binding domains. PSMT1 undergoes an inward rotation of approximately 10 degrees of the SAM binding domain upon substrate binding. This results in an 8 Å closure of the active site cleft. The acceptor substrate binds in a hydrophobic cleft, held in place by two hydrogen bonds via the hydroxyl groups at positions 2 and 9 of scoulerine. Further analysis of the plant O-methyltransferase catalytic dyad has been carried out by site-directed mutagenesis and X-ray diffraction studies. Subunit cooperativity has also been investigated with the proposal of two regulatory features. Here I present one of the highest resolution closed conformation structure of a plant O-methyltransferase to date. This provides the template for our understanding of, and future engineering substrate specificity in plant methyltransferases; an important class of enzymes in secondary metabolism.
Supervisor: Graham, Ian A. ; Davies, Gideon J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.739930  DOI: Not available
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