2-keto-3-deoxy-6-phosphogluconate

Mavridis, I.M., et al. Structure of 2-keto-3-deoxy-6-phosphogluconate aldolase at 2.8 A resolution. 

Fong, S., Machajewski, T.D., Mak, C.C. and Wong, C.-H. (2000) Directed evolution of D-2-keto-3-deoxy-6-phosphogluconate aldolase to new variants for the efficient synthesis of d- and L-sugars. Chemistry Biology, 7, 873-883. 

Aldolase, 2-keto-3-deoxy-6-phosphogluconate (Mavridis and Tulin-sky, 1976 Richardson, 1979)... 

The 2-keto-3-deoxy-6-phosphogluconate aldolase (KDPG aldolase EC 4.1.2.14) catalyzes the cleavage of the dehydration product of 6-phosphogluconate, (KDPG), into glyceraldehyde-3-phosphate and pyruvate in the Entner-Doudoroff pathway (Scheme 7, R = P03H2). This... 

H. P. Meloche, J. M. Ingram, and W. A. Wood, 2-Keto-3-deoxy-6-phosphogluconic aldolase (crystalline). Methods Enzymol. 9 520 (1966). 

Wood WA (1972) 2-Keto-3-deoxy-6-phosphogluconic and Related Aldolases. In Boyer PD (ed) The Enzymes, 3rd ed. Academic, New York, vol. 7, p 281... 

L. Pocivavsek, C. A. Fierke, E. J. Toone, and J. H. Naismith, Directed evolution of a new catalytic site in 2-keto-3-deoxy-6-phosphogluconate aldolase from Escherichia coli, Structure 2001, 9, 1-10. 

The carbon-carbon forming ability of aldolases has been limited in part by their narrow substrate utilization. Site-directed mutagenesis of various enzymes to alter their specificity has most often not produced the desired effect. Directed evolution approaches have furnished novel activities through multiple mutations of residues involved in recognition in no instance has a key catalytic residue been altered while activity is retained. Random mutagenesis resulted in a double mutant of E. coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase with reduced but measurable enzyme activity and a synthetically useful substrate profile (Wymer, 2001). 

M. C. Shelton, I. C. Cotterill, S. T. A. Novak, R. M. Poonawala, S. Sudarshan, and E. J. Toone, 2-Keto-3-deoxy-6-phosphogluconate aldolases as catalysts for stereocon-trolled carbon-carbon bond formation, J. Am. Chem. Soc., 118 (1996) 2117-2125. 

The product, 2-keto-3-deoxy-6-phosphogluconate, was separated by chromatography at room temperature and a flow rate of 1 mL/min on a Dionex CarboPac PA-1 column (4 mm x 250 mm). The mobile phase was composed of 24 mM NaOH and 300 mM sodium acetate for 5 minutes, followed by a linear gradient to 700 mM sodium acetate in 10 minutes. A linear gradient back to the initial conditions was run in 5 minutes. Pulsed amperometric detection was used with a pulse train consisting of a 480 ms detection pulse at +80 mV, followed by pulses of 120 ms at +600 mV and 60 ms at -600 mV. 

Figure 9.80 Representative chromatographs of Entner-Doudoroff metabolites, (a) 6-Phosphogluconate dehydratase-catalyzed formation of 2-keto-3-deoxy-6-phosphogluconate (12.51 min). (b) Blank run for 6-phosphogluconate dehydratase-catalyzed formation of 2-keto-3-deoxy-6-phosphogluconate, (KDPG) 6-phosphogluconate omitted from assay. (From Taha and Deits, 1994.)...

Enzyme used in the assays was purified from Azotobacter vinelandii A modification of the assay was used to follow the approach to equilibrium of the 2-keto-3-deoxy-6-phosphogluconate aldolase reaction. 

As mentioned, the mildness of NaBHaCN (coupled with its effectiveness and stability in aqueous media) has attracted considerable interest for applications in biochemical areas. Examples include the trapping of suspected imine intermediates produced in enzyme (mitochondrial monoamine oxidase) inactivation by amines, the establishment by reduction of the positions of imine-forming amines in 2-keto-3-deoxy-6-phosphogluconate aldolase, and the transfer labeling of methionyl-tRNA synthetase and methionyl-tRNA transformalase by treatment with periodate-treated tRNA. In fact, most biochemical applications of NaBHaCN have utilized in situ imine formation-reduction (i.e. reductive amination) conditions and will be further discussed in Section 1.2.2.3.1. 

Lebioda, L., Hatada, M. H., Tulinsky, A., and Mavridis, I. Comparison of the folding of 2-keto-3-deoxy-6-phosphogluconate aldolase, triose phosphate iso-mercise and pyruvate kincise. Implications in molecular evolution. J. Molec. Biol. 162, 445-458 (1982). 

Keto-3-deoxy-6-phosphogluconic and Related Aldolases W. A. Wood... 

Some initial studies have been made on KDPG aldolase (EC 4.1.2.14) which in vivo catalyzes the reversible condensation of the pyruvate with glyceraldehyde 3-phosphate (25) to form 2-keto-3-deoxy-6-phosphogluconate (KDPG - 26) (Scheme 9). The enzyme was found to accept a number of unnatural aldehydes as electrophiles. It was applied for the synthesis of KDG from glyceraldehyde [66]. 

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