Biosynthetic enzymes expression cloning

The study of Bi2 systems has involved research in inorganic/organometallic syntheses, bioorganic chemistry, biosynthetic chemistry, electrochemistry, theoretical chemistry, and spectroscopy in addition to the typical studies associated with enzymes (kinetics, cloning and expression, etc.). Of particular note, since the B l2 compounds are among the most complicated natural products, they often serve as a venue for studies with new methods and for the extension of methods from small molecules to macromolecules. X-ray diffraction methods have revealed that in both known types of human enzymes, the coenzyme binds in the base-off form with a protein imidazole side chain bound to the Co. 

Mammalian cDNAs encoding the two isoforms of PS synthase were subsequently cloned. The amino acid sequences of PS synthase-1 and PS synthase-2 are -30% identical and each protein is predicted to contain multiple membrane-spanning domains. As is the case for many lipid biosynthetic enzymes, the serine-exchange activity in mammalian cells is located on microsomal membranes. However, the majority of this activity is not in the bulk of the ER but is highly enriched in MAM, a domain of the ER that has been implicated in the import of PS into mitochondria (J.E. Vance, 2000) (Chapter 16). Although little is known about the active site of either PS synthase, several amino acids that are crucial for serine-exchange activity have been identified (M. Nishijima, 2004). Over-expression of PS synthase-2 in PS synthase-1-deficient CHO cells eliminated the requirement for exogenously added PS. Thus, in CHO cells PS synthase-2 can substitute for PS synthase-1. 

In the past decade all the heparin biosynthetic enzymes have been isolated, cloned and expressed. These enzymes, while still incompletely characterized, have recently been used to prepare the AT-binding site. This technical achievement resulted in only 1 pg of AT-binding site and is also limited to the preparation of only naturally occurring structures. A new chemoenzymatic synthetic approach, such as that being developed in our laboratories, affording multimilligram amounts of GAG oligosaccharides with a variety of natural and unnatural structures is needed. 

The greatest limitation of functional cloning is the design of effective enzyme assays that can rapidly assess the function of thousands of clones. Unfortunately, relatively few metabolic enzymes yield a colored product that can be easily detected. Furthermore, many biosynthetic enzymes act on unavailable, miknown or imstable biosynthetic intermediates that are not suitable for an in vitro enzyme assay. The plant enzymes must also be expressed in active form in E. coli or yeast for accurate functional assessment, and some plant proteins can only be functionally expressed in insect or plant cells, which are not amenable for this functional expression strategy. Finally, expression cloning requires exceptionally high quality cDNA that consists of full-length clones. 

Capsaicinoids are synthesized by the condensation of vanillylamine with a short chain branched fatty acyl CoA. A schematic of this pathway is presented in Fig. 8.4. Evidence to support this pathway includes radiotracer studies, determination of enzyme activities, and the abundance of intermediates as a function of fruit development [51, 52, 57-63], Differential expression approaches have been used to isolate cDNA forms of biosynthetic genes [64-66], As this approach worked to corroborate several steps on the pathway, Mazourek et al. [67] used Arabidopsis sequences to design primers to clone the missing steps from a cDNA library. They have expanded the schema to include the biosynthesis of the key precursors phenylalanine and leucine, valine and isoleucine. Prior to this study it was not clear how the vanillin was produced, and thus the identification of candidate transcripts on the lignin pathway for the conversion of coumarate to feruloyl-CoA and the subsequent conversion to vanillin provide key tools to further test this proposed pathway. 

WaUaart TE, Bouwmeester HJ, Hille J, Poppinga L, Maijers NCA (2001) Amorpha-4, 11-diene synthase cloning and functional expression of a key enzyme in the biosynthetic pathway of the novel antimalarial drug artemisinin. Planta 212 460-465... 

Lacombe, E., Hawkins, S., Van Doorsselaere, J., Piquemal, J., Goffher, D., Poeydomenge, O., Boudet, A.-M., and Grima-Pettenati, J., 1997, Cinnamoyl Co A reductase, the first committed enzyme of the lignin branch biosynthetic pathway cloning, expression and phylogenetic relationships, Plant J. 11 429-441. 

Biosynthetic research relating to the isoquinoline family was extremely successful, with such important members as morphine [3, 14], codeine [3, 15] or berberine [3, 14,16-18]. Extensive efforts have provided details pertaining to multiple sets of enzymes participating in the biosynthesis of the alkaloids above, in many cases with the help of plant cell suspension culture techniques. Since 1988, when the breakthrough in cloning of cDNA from alkaloid biosynthesis occurred [19, 20], a significant number of enzymes known from the indoles and isoquinolines biosynthesis have been isolated, their biochemical properties described and the majority of their corresponding cDNAs cloned and functionally over-expressed in non-plant hosts such as Escherichia coli, yeast or insect cells. 

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