University of Leicester
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Mechanistic and structural investigations of DDG aldolase

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posted on 2014-12-15, 10:35 authored by Nicholas Charles. Blackwell
Carbon-Carbon bond formation is an essential reaction in organic chemistry and the use of aldolase enzymes for the stereochemical control of such reactions is an attractive alternative to conventional chemical methods. A type IIB aldolase (utilises pyruvate as a nucleophilic substrate, metal-ion requiring enzyme), 2-dehydro-3-deoxy-D-galactarate (DDG) aldolase from Escherichia coli, EC, a member of the glucarate and galactarate metabolic pathways where it is responsible for the in vitro cleavage of 2-dehydro-3-deoxy-D-glucarate to pyruvate and tartronic semialdehyde, was investigated for its application to such processes. This study describes the purification of the enzyme from E. coli NM522 and subsequent cloning into the pT77 RNA polymerase promoter expression system, as well as detailed analyses of both the cleavage and condensation reactions for the aldolase, under a variety of different conditions including buffer type, ionic strength, pH, substrate excess and co-solvents. Investigations into the affinity of DDG aldolase for a wide range of aldehydes have established that the enzyme only forms condensation compounds with configuration R at the new stereogenic centre and shows no selectivity towards the configuration at positions C-2 and C-3 of the substrate. DDG aldolase also displays a greater tolerance to electrophilic substrate type than that currently reported for the type 1 aldolases, notably fructose-1, 6-bisphosphate aldolase. Where applicable, a selection of novel condensation compounds have been purified and characterised and a suitable method of large scale production of said compounds presented. Crystal structures of the recombinant and seleno-methionine isoforms are also presented, establishing that DDG aldolase is a homo-hexamer of subunit molecular weight 27.4 kDa, with the promoter displaying a modified (/)8 barrel fold. Analysis of the DDG aldolase structure and kinetic investigations suggests that the enzyme displays a novel aldolase mechanism.


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University of Leicester

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  • Doctoral

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  • PhD



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