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

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

Interaction of the Agrobacterium tumefaciens virulence protein VirD2 with histones.

Agrobacterium tumefaciens is a Gram-negative soil bacterium that genetically transforms plants and, under laboratory conditions, also transforms non-plant organisms, such as fungi and yeasts. During the transformation process a piece of ssDNA (T-strand) is transferred into the host cells via a type IV secretion system. The VirD2 relaxase protein, which is covalently attached at the 5' end of the T-strand through Tyr29, mediates nuclear entry as it contains a nuclear localization sequence. How the T-strand reaches the chromatin and becomes integrated in the chromosomal DNA is still far from clear. Here, we investigated whether VirD2 binds to histone proteins in the yeast Saccharomyces cerevisiae. Using immobilized GFP-VirD2 and in vitro synthesized His6-tagged S. cerevisiae proteins, interactions between VirD2 and the histones H2A, H2B, H3 and H4 were revealed. In vivo, these interactions were confirmed by bimolecular fluorescence complementation experiments. After co-cultivation of Agrobacterium strains expressing VirD2 tagged with a fragment of the yellow fluorescent protein analogue Venus with yeast strains expressing histone H2A or H2B tagged with the complementary part of Venus, fluorescence was detected in dot-shaped structures in the recipient yeast cells. The results indicated that VirD2 was transferred from Agrobacterium to yeast cells and that it interacted with histones in the host cell, and thus may help direct the T-DNA (transferred DNA) to the chromatin as a prelude to integration into the host chromosomal DNA.Plant science

DHAP-dependent aldolases from (hyper)thermophiles: biochemistry and applications

Generating new carbon-carbon (C-C) bonds in an enantioselective way is one of the big challenges in organic synthesis. Aldolases are a natural tool for stereoselective C-C bond formation in a green and sustainable way. This review will focus on thermophilic aldolases in general and on dihydroxyacetone phosphate-dependent aldolases in particular. Biochemical properties and applications for synthesis of rare sugars and carbohydrates will be discusse

Substrate Specificity and Stereoselectivity of Two Sulfolobus 2-Keto-3-deoxygluconate Aldolases towards Azido-Substituted Aldehydes

The 2-keto-3-deoxygluconate aldolases (KDGAs) isolated from Sulfolobus species convert pyruvate and glyceraldehyde reversibly into 2-keto-3-deoxygluconate and -galactonate. As a result of their high thermostability and activity on nonphosphorylated substrates, KDGA enzymes have potential as biocatalysts for the production of building blocks for fine chemical and pharmaceutical applications. Up to now, wild-type enzymes have only shown moderate stereocontrol for their natural reaction. However, if a set of azido-functionalized aldehydes were applied as alternative acceptors in the reaction with pyruvate, the stereoselectivity was strongly increased to give enantiomeric or diastereomeric excess values up to 97¿%. The Sulfolobus acidocaldarius KDGA displayed a higher stereoselectivity than Sulfolobus solfataricus KDGA for all tested reactions. The azido-containing products are useful chiral intermediates in the synthesis of nitrogen heterocycles

Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis

Dihydrodipicolinate synthase (DHDPS) catalyses the first reaction of the (S)-lysine biosynthesis pathway in bacteria and plants. The hypothetical gene for dihydrodipicolinate synthase (dapA) of Thermoanaerobacter tengcongensis was found in a cluster containing several genes of the diaminopimelate lysine¿synthesis pathway. The dapA gene was cloned in Escherichia coli, DHDPS was subsequently produced and purified to homogeneity. The T. tengcongensis DHDPS was found to be thermostable (T 0.5 = 3 h at 90°C). The specific condensation of pyruvate and (S)-aspartate-ß -semialdehyde was catalyzed optimally at 80°C at pH 8.0. Enzyme kinetics were determined at 60°C, as close as possible to in vivo conditions. The established kinetic parameters were in the same range as for example E. coli dihydrodipicolinate synthase. The specific activity of the T. tengcongensis DHDPS was relatively high even at 30°C. Like most dihydrodipicolinate synthases known at present, the DHDPS of T. tengcongensis seems to be a tetramer. A structural model reveals that the active site is well conserved. The binding site of the allosteric inhibitor lysine appears not to be conserved, which agrees with the fact that the DHDPS of T. tengcongensis is not inhibited by lysine under physiological conditions

Substrate Specificity and Stereoselectivity of Two Sulfolobus 2-Keto-3-deoxygluconate Aldolases towards Azido-Substituted Aldehydes

The 2-keto-3-deoxygluconate aldolases (KDGAs) isolated from Sulfolobus species convert pyruvate and glyceraldehyde reversibly into 2-keto-3-deoxygluconate and -galactonate. As a result of their high thermostability and activity on nonphosphorylated substrates, KDGA enzymes have potential as biocatalysts for the production of building blocks for fine chemical and pharmaceutical applications. Up to now, wild-type enzymes have only shown moderate stereocontrol for their natural reaction. However, if a set of azido-functionalized aldehydes were applied as alternative acceptors in the reaction with pyruvate, the stereoselectivity was strongly increased to give enantiomeric or diastereomeric excess values up to 97¿%. The Sulfolobus acidocaldarius KDGA displayed a higher stereoselectivity than Sulfolobus solfataricus KDGA for all tested reactions. The azido-containing products are useful chiral intermediates in the synthesis of nitrogen heterocycles

(Hyper)thermophilic enzymes : Production and purification

The discovery of thermophilic and hyperthermophilic microorganisms, thriving at environmental temperatures near or above 100 °C, has revolutionized our ideas about the upper temperature limit at which life can exist. The characterization of (hyper)thermostable proteins has broadened our understanding and presented new opportunities for solving one of the most challenging problems in biophysics: how are structural stability and biological function maintained at high temperatures where “normal” proteins undergo dramatic structural changes? In our laboratory, we have purified and studied many thermostable and hyperthermostable proteins in an attempt to determine the molecular basis of heat stability. Here, we present methods to express such proteins and enzymes in E. coli and provide a general protocol for overproduction and purification. The ability to produce enzymes that retain their stability and activity at elevated temperatures creates exciting opportunities for a wide range of biocatalytic applications.</p