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Characterization and engineering of thermophilic aldolases : synthesizing nitrogen-heterocycles in biosynthetic routes

  • Wolterink-van Loo, S.
Publication Date
Jan 01, 2009
Wageningen University and Researchcenter Publications
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Aldolases are enzymes that catalyze reactions in both degradation and biosynthetic pathways in vivo and have been discovered in all domains of life. they. An interesting property of aldolases is that they can synthesize carbon-carbon bonds, generating a new stereogenic centre. As enzymes are generally stereo-selective, they can be useful in generating pure stereo-isomers as building blocks for pharmaceuticals and fine chemicals. Another requirement for application of enzymes is a long shelf life and stability. Enzymes from extremophiles, such as (hyper)thermophilic micro-organisms are usually very stable, e.g. at high temperatures and in different organic solvents. The bacterial dihydrodipicolinate synthase (DHDPS) and the crenarchaeal 2-keto-3-deoxygluconate aldolase (KDGA) are two pyruvate-dependent aldolases with activity on non-phosphorylated (cheap) substrates. Their potential for biotechnological application, -in synthesizing nitrogen-heterocycles in biosynthetic routes-, has been explored in this thesis. DHDPS from the bacterium Thermoanaerobacter tengcongensis (TteDHDPS) was produced using E. coli. This thermostable TteDHDPS appeared very specific for the (S)-ASA aldehyde substrate and therefore was not investigated further. Three Sulfolobus KDGAs have been produced in E. coli as well. Purified protein of S. acidocaldarius KDGA (SacKDGA) was crystallized, and the 3-dimensional structure was solved, using the S. solfataricus KDGA (SsoKDGA) structure. The KDGAs proved to have a remarkable broad substrate specificity regarding the aldehyde acceptor: aldehydes with 2-5 carbon atoms were accepted, and additionally azido-substituted aldehydes were readily accepted as well. This observed broad substrate specificity correlates well with the rather spacious hydrophilic binding site that was found in the 3-dimensional structure of KDGA. Using random optimization techniques SacKDGA was improved for low-temperature catalysis. A single mutation V193A, near the active site, increased the activity of the aldolase condensation reaction of glyceraldehyde and pyruvate 3 times at 50°C. Thorough characterization of products of SsoKDGA and SacKDGA reactions, using pyruvate and different aldehydes showed that SacKDGA was much more specific towards synthesis of the S-enantiomer, using different aldehyde substrates. Furthermore, using the 3-dimensional structure of KDGA an attempt was made to increase the stereoselectivity of this enzyme towards synthesis of 5-azido-4(S)-hydroxy-2-oxopentanoic acid. Based on computer predictions on binding of 5-azido-4(S)-hydroxy-2-oxopentanoic acid, up to three amino acid changes were introduced and all mutants were characterized separately. The specificity of the enzyme had shifted from the glyceraldehyde substrate towards the 2-azidoacetaldehyde substrate, but unfortunately the effect on stereospecificity was quite small. Nevertheless, using aldolase-based biocatalysis in combination with relatively simple follow-up chemistry, we were able to produce the nitrogen heterocycles 4-hydroxyproline, 4-hydroxy-5-methylproline and 2-carboxy-4-hydroxypiperidine.

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