Investigation of a Possible Multi-enzyme Complex Involved in Nicotine Biosynthesis in Roots of Tobacco (Nicotiana tabacum)
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N-methylputrescine oxidase (MPO) is a member of the diamine oxidase (DAO) class of enzymes believed to be responsible for synthesis of the alkaloid nicotine in the roots of Nicotiana tabacum (Mizusaki et al., 1972). A purportedly pure MPO protein from tobacco root culture extracts was used to generate immune antiserum in rabbits (McLauchlan et al., 1993). In an attempt to clone a cDNA encoding MPO, we used this antiserum to screen a tobacco cDNA expression library. Unexpectedly, two previously unreported genes with strong homology to members of a gene family encoding S-adenosylhomocysteine hydrolase (SAHH) in N. sylvestris and a gene encoding SAHH in N. tabacum were cloned instead. SAHH is an enzyme of the S-adenosylmethionine (SAM) recycling pathway, which also includes SAM synthetase (SAMS) and methionine synthase (MS). These results led to the hypothesis of a multi-enzyme complex, or metabolon, of at least one member of the nicotine biosynthesis pathway, i.e., MPO, and at least one member of the SAM recycling pathway, i.e., SAHH, during nicotine biosynthesis. Metabolons are stable noncovalent complexes in cells that ensure sufficient passage of the product of one enzyme reaction to the next enzyme in the pathway via a "channel" without equilibrating with the bulk solution (OvÃ¡di, 1991). My research employed co-immunoprecipitation studies to determine if other SAM recycling enzymes are associated in a complex with MPO and SAHH, as well as Northern and Western blot analyses to determine if the genes encoding SAM recycling pathway enzymes are coordinately regulated during nicotine biosynthesis. Our results indicate that nicotine biosynthesis-inducing conditions result in differential mRNA accumulation patterns of the three enzymes of the SAM recycling pathway, although to different extents. However, protein levels of SAM recycling pathway members do not appear to reflect the differential mRNA accumulation patterns. We have firmly established an association of SAHH and an enzyme with DAO activity, purportedly MPO. If the enzyme is proven to be MPO, then our data would constitute the first documentation of an alkaloid metabolon. Finally, using a degenerate primer PCR approach, we have cloned a 986-bp gene fragment with homology to copper amine oxidases, the class to which MPO belongs.
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