Introduction cofactor requirements

Flavonol synthase (FLS E.C.l.14.11.23) catalyzes the committed step in the production of fiavonols by introduction of a double bond between C2 and C3 of the corresponding dihydroflavonols. Like E3H, ELS has been described as a 2-oxoglutatarate-dependent dioxygenase based on its cofactor requirements for 2-oxoglutarate, Fe, and ascorbate. FLS was initially identified in enzyme preparations from illuminated parsley cell suspension cultures [67]. Subsequently, FLS was characterized from the flower buds of Matthiola incana and carnation (Dianthus caryophyllus L.), and it was suggested that there was regulation between flavonol and anthocyanidin biosynthesis [83, 84]. 

Each chapter is subdivided into sections beginning with a short introduction or general description, which gives a brief overview of the subject. Then functional, structural, and genetic aspects of each enzyme are described, with examples of typical applications. Functional aspects include (1) reaction conditions (optimal or recommended reaction conditions, kinetic parameters, cofactor requirements, and inhibitors/inactivators) (2) activity assay and unit definition (3) substrate specificity and (4) catalytic mechanism. The section on enzyme functions should enable the reader to predict the possible outcome of a reaction under a given condition and provide the basis for which the activity of an enzyme can be optimized or fine tuned to obtain the desired results. 

The majority of hydroxylations are performed with whole microorganisms. The best procedure for the introduction of hydroxy groups would be the use of isolated specific hydroxylases. However, it has not proven feasible to use cell-free hydroxylases as the enzymes involved are usually membrane-bound and thus not easily isolated and purified. Furthermore, the hydrox-ylation reaction requires the addition of NADH, an expensive cofactor. 

The enzyme is attached to the sensing electrode itself. Solution flow is shown in Figure 2C only to suggest a method of analyte introduction to the biosensor. This system does not require flow past the enzyme and detector but instead relies on diffusion of substrate to the enzyme, and diffusion of the enzymatically generated reduced cofactor fi om the inunobilized enzyme to the working electrode. As will be shown, inuno-biUzation of enzymes on the electrode surface often reduces the detector efficiency because both enzyme conversion efficiency and diffusion of analytes can limit the time response of such a system. This type of system is amenable to the immobilization of the cofactor as well (e.g., wired enzyme electrodes [5-7], which use intrinsic FAD/FADHj as cofactors). 

The stereospecific introduction of a Z-double bond (Kcurs by the abstraction of two vicinal pro-/ hydrogens atoms at C-9 and C-10 in a thioester (Fig. 3.3). In plants, R = ACP, while in animals and fungi, the thioester is activated as CoA. This reaction is catalyzed by stearoyl-ACP A -desaturase in plants and stearoyl-CoA A -desaturase in animals and fungi [1]. Oxygen and either NADPH or NADH as cofactors are required for both types of desaturases. 

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