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A major pathway of tyramine and dopamine synthesis in insects is through the decarboxylation of tyrosine and DOPA, respectively. Although tyrosine decarboxylase (TDC) has been mentioned in some reports, it has never been critically analyzed. The high sequence identity shared by tyrosine decarboxylase and DOPA decarboxylase in insects, and the similar structures of the substrates, tyrosine and DOPA, raise the possibility that both tyrosine decarboxylase and DOPA decarboxylase (DDC) have activities to tyrosine and DOPA. In this study, after tyrosine decarboxylase and DOPA decarboxylase enzymes of Anopheles gambiae were expressed, their substrate specificities and biochemical properties were critically analyzed. My results provide clear biochemical evidence establishing that the mosquito tyrosine decarboxylase functions primarily on the production of tyramine with low activity to DOPA. In contrast, mosquito DOPA decarboxylase is highly specific to DOPA with essentially no activity to tyrosine. / Master of Science in Life Sciences

Abstract Rhodiola rosea is a medicinal plant, mainly used in Asia and Scandinavia. It is characterized as an adaptogen and is reported to have many pharmacological properties, which are ascribed to the glycosides of cinnamyl alcohol and tyrosol. As natural habitats are already overharvested and the cultivation of this plant needs 4–6 years, the production of the pharmacologically important compounds in in vitro cultures could be an alternative. In the work presented here, the production of these glycosides in compact callus aggregate cultures of roseroot was addressed. Biotransformation of exogenously added cinnamylalcohol and tyrosol was studied. Glucosylation of the precursors yielded high amounts of rosin and salidroside and low amounts of rosavin. During the course of this work, four new glycosides of cinnamyl alcohol were found and identified. The optimal concentration of the precursors and the time needed for the biotransformation was also determined. For enhancing the biotransformation rate, glucose was added to the culture medium alongside with sucrose, which doubled the production of cinnamyl alcohol glycosides but did not affect the production of salidroside. A pilot experiment using air-lift bioreactor was performed. A cDNA fragment encoding tyrosine decarboxylase was isolated and described. The expression of this gene was analysed in the leaves and roots of two chemotypes. The results demonstrate the important role of tyrosine decarboxylase in the production of salidroside. The results revealed production of the pharmacologically important glycosides of Rhodiola rosea; however the successful pilot bioreactor experiment remains to be scaled-up. New information was obtained on the biosynthesis of salidroside, which substantiate the metabolic engineering of roseroot.

Plant aromatic amino acid decarboxylases (AAADs) are a group of economically important enzymes categorically joined through their pyridoxal 5'-phosphate (PLP) dependence and sequence homology. Extensive evolutionary divergence of this enzyme family has resulted in a selection of enzymes with stringent aromatic amino acid substrate specificities. Variations in substrate specificities enable individual enzymes to catalyze key reactions in a diverse set of pathways impacting the synthesis of monoterpenoid indole alkaloids (including the pharmacologically active vinblastine and quinine), benzylisoquinoline alkaloids (including the pharmacologically active papaverine, codeine, morphine, and sanguinarine), and antioxidant and chemotherapeutic amides. Recent studies of plant AAAD proteins demonstrated that in addition to the typical decarboxylation enzymes, some annotated plant AAAD proteins are actually aromatic acetaldehyde synthases (AASs). These AASs catalyze a decarboxylation-oxidative deamination process of aromatic amino acids, leading to the production of aromatic acetaldehydes rather than the AAAD derived arylalkylamines. Research has implicated that plant AAS enzymes are involved in the production of volatile flower scents, floral attractants, and defensive phenolic acetaldehyde secondary metabolites. Historically, the structural elements responsible for differentiating plant AAAD substrate specificity and activity have been difficult to identify due to strong AAAD and AAS inter-enzyme homology. Through extensive bioinformatic analysis and experimental verification of plant AAADs, we have determined some structural elements unique to given types of AAADs. This document highlights structural components apparently responsible for the differentiation of activity and substrate specificity. In addition to producing primary sequence identifiers capable of AAAD activity and substrate specificity differentiation, this work has also demonstrated applications of AAAD enzyme engineering and novel activity identification. / Ph. D.