Mechanistic studies on norcoclaurine synthase of benzylisoquinoline alkaloid biosynthesis: an enzymatic Pictet-Spengler reaction.
Norcoclaurine synthase catalyzes an asymmetric Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde to give (S)-norcoclaurine. This is the first committed step in the biosynthesis of the benzylisoquinoline alkaloids that include morphine and codeine. In this work, the gene encoding for the Thalictrum flavum norcoclaurine synthase is highly overexpressed in Escherichia coli and the resulting His-tagged recombinant enzyme is purified for the first time. A continuous assay based on circular dichroism spectroscopy is developed and used to monitor the kinetics of the enzymatic reaction. Dopamine analogues bearing a methoxy or hydrogen substituent in place of the C-1 phenolic group were readily accepted by the enzyme whereas those bearing the same substituents at C-2 were not. This supports a mechanism involving a two-step cyclization of the putative iminium ion intermediate that does not proceed via a spirocyclic intermediate. The reaction of [3,5,6-2H]dopamine was found to be slowed by a kinetic isotope effect of 1.7 +/- 0.1 on the value of kcat/KM. This is interpreted as showing that the deprotonation step causing rearomatization is partially rate determining in the overall reaction.
Luk
Louis Y P
LY
Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1.
Bunn
Shannon
S
Liscombe
David K
DK
Facchini
Peter J
PJ
Tanner
Martin E
ME
D000470:Alkaloids / Q000096:biosynthesis*; D044182:Benzylisoquinolines / Q000378:metabolism*; D019731:Carbon-Nitrogen Ligases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D002942:Circular Dichroism; D003061:Codeine / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D004298:Dopamine / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism*; D007700:Kinetics; D015394:Molecular Structure; D009020:Morphine / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification*; D031949:Thalictrum / Q000201:enzymology*
D000470:Alkaloids; D044182:Benzylisoquinolines; D011993:Recombinant Fusion Proteins; D009020:Morphine; D019731:Carbon-Nitrogen Ligases; C450500:norcoclaurine synthase; D003061:Codeine; D004298:Dopamine