Cofactors, introduction

Cofactor introduction at a unique active site lysine of aldolase antibodies. 

Introduction of heme residues and different artificial receptors in protein molecules in chemical modification of structures and functions of proteins by the cofactor reconstruction method 99Ef0539. 

Step by step introduction of more effective ways of handling cytoplasmic chemistry, using coenzymes and special metal cofactors, e.g. haem (Fe), vitamin B12(Co), F-430(Ni), chlorophyll(Mg) and Moco(Mo). 

Introductions to enzyme kinetics and bioenergetics are given with explanations of key terms such as Km and Vmax coenzymes, cofactors and inhibitors typical metabolic reactions free energy exergonic and endergonic reactions, catabolism and anabolism. 

Biosynthetic production of thymidine is overall a complex process combining the controlled introduction of a novel biotransformation step into a biological system with selective enhancement or knock-out of a series of existing metabolic steps. Metabolic engineering to enhance cofactor recycling at both ribonucleotide reduction and dUMP methylation steps has important parallels in other systems, as whole-cell biotransformations are frequently employed as a means to supply, in situ, high-cost and usually labile cofactors. 

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]. 

The condensation step is catalyzed by the glycosyltransferase SpcF. Further enzyme-catalyzed oxidation is probably needed for the introduction of the hemiketal linkage between cyclitol and sugar units. The SpcY enzyme, which has a similar counterpart, HygY, among the /lyg-cluster encoded proteins (see Section 2.2.4.3.1), is a candidate enzyme for this reaction. SpcY is a member of the radical SAM superfamily of proteins and relatives of SpcY have been found before all in connection with molybdenum-cofactor biosynthesis but to our knowledge no details of the mechanism involved is known for those. 

In addition, cofactor engineering has been used to deliberately modify the intracellular NADH/NAD+ ratio that plays a predominant role in controlling the Lactococcus lactis fermentation pattern. The introduction of the nox gene, which codes for a NADH oxidase (NOX) that converts molecular oxygen to water at the expense of NADH, to a strain with an inactivated copy of the aldB gene for a-acetolactate decarboxylase led to the efficient metabolism of the na-... 

A different approach for utilization of the photoproducts in chemical routes involves the introduction of natural enzymes as catalysts in the photochemical system. In nature, dihydronicotinamide adenine dinucleotide (NADH) and dihydronicotinamide dinucleotide phosphate (NADPH) participate as reducing cofactors in a variety of enzymatic reduction processes. Thus, the development of photochemical NADH and NADPH regeneration cycles is anticipated to allow a variety of reduction processes by inclusion of substrate specific NAD(P)H dependent enzymes. 

The corresponding high-valent intermediate in the assembly of the di-iron(III) center-tyrosyl radical cofactor of RNR R2 has also been identified by Stubbe and coworkers and designated as X [86,89], This intermediate decays to the (p,-oxo)diiron(III) form at a rate commensurate with the appearance of the tyrosyl radical. Intermediate X, formally Fe(III)Fe(IV), exhibits an isotropic S = 1/2 spin EPR signal near g = 2, which is split by the introduction of 57Fe and broadened by 1702 in the assembly reaction. These observations as well as Mossbauer results show that the unpaired spin must be associated with the diiron center [88,89],... 

Regardless of the expression of the receptor and cofactors, the reporter gene must be introduced to the cells. There are several options for the introduction of the reporter gene construct into the cells including chemical-based transfection (CaClj), lipid-based transfection, and electroporation. 

There also are significant differences between the enzymes. First, there is the different site of introduction of the double bond. Then there is the preferred cofactor both will accept NADPH or NADH, but the A9 enzyme prefers the former and the All enzyme the latter. Both are sensitive to cyanide, but the All is about 50 times less sensitive. The pH optimum of the A9 enzyme is somewhat more acidic (7.8-7.2). Finally, there is the tissue location. The A9 enzyme is generally present throughout the insect, while the All enzyme is localized in the pheromone gland (Zl). ... 

Fig. 34 Enzymatic reduction in biphasic media. Ketones are reduced enantioselective to the corresponding (S)-alcohols by ADH from Rhodococcus erythropolis. Regeneration of the cofactor NADH is carried out by FDH from C. boidinii. The introduction of a biphasic system allows higher substrate concentrations...

Most of these enzymatic ways to regenerate nicotinamide cofactors have been successfully established in a multitude of synthetic processes. The introduction of organic-water two-phase systems seems to be a useful method to overcome problems with the low solubility of many organic substrates. Bioconversions using whole cells have been well investigated because they show some decisive advantages like increased enzyme stability. The use of recombinant DNA techniques offers a wide field of application. Tailor-made cells can be created to perform whole cell biotransformations efficiently. 

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