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Dirk Franke

Publications and source records attributed to Dirk Franke.

9 recordsLinked to original sources
PubMed2005

Directed evolution of D-sialic acid aldolase to L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase.

An efficient L-3-deoxy-manno-2-octulosonic acid (L-KDO) aldolase was created by directed evolution from the Escherichia coli D-Neu5Ac (N-acetylneuraminic acid, D-sialic acid) aldolase. Five rounds of error-prone PCR and iterative screening were performed with sampling of 10(3) colonies per round. The specificity constant (kcat/Km) of the unnatural sugar L-KDO is improved to a level equivalent to the wild-type D-sialic acid aldolase for its natural substrate, D-Neu5Ac. The final evolved enzyme exhibits a >1,000-fold improved ratio of the specificity constant [kcat/Km (L-KDO)]/[kcat/Km (D-sialic acid)]. The protein sequence of the evolved aldolase showed eight amino acid changes from the native enzyme, with all of the observed changes occurring outside of the active site. Our effort demonstrates that an enzyme can be rapidly altered to accept enantiomeric substrates with screening of a small population of colonies iteratively toward the target substrate with improved catalytic efficiency. This work provides a method for the synthesis of enantiomeric sugars and for the study of enantiomeric catalysis affected by remote mutations.

Amino Acids↗
PubMed2005

A new strategy for glycoprotein synthesis: ligation of synthetic glycopeptides with truncated proteins expressed in E. coli as TEV protease cleavable fusion protein.

We report here the use of TEV protease cleavable fusion proteins to produce glycosylated bioactive peptides and proteins. Bacterial expression was utilized to produce two fusion proteins, GPRT-C37-H6 and His-tagged interleukin-2 (amino acids 6-133), which when cleaved by the tobacco etch virus NIa protease (TEV protease) to generate HIV entry inhibitor peptide C37-H6 and a truncated version of the cytokine interleukin-2, both containing N-terminal cysteines. The N-terminal cysteine containing C37-H6 and truncated interleukin-2 were then joined to a synthetic glycopeptide thioester utilizing native chemical ligation under nondenaturing and denaturing conditions, respectively. The ligations of the glycopeptide to the C37-H6 peptide and the truncated interleukin-2 protein both proceeded in high yield, though the size, and physical properties of the two polypeptides differ greatly.

Base Sequence↗
PubMed2003

(S,S)-2,3-Dihydroxy-2,3-dihydrobenzoic acid: microbial access with engineered cells of Escherichia coli and application as starting material in natural-product synthesis.

Cyclohexadiene-trans-5,6-diols such as (S,S)-2,3-dihydroxy-2,3-dihydrobenzoic acid (2,3-trans-CHD) have been shown to be of importance as chiral starting materials for the syntheses of bioactive substances, especially for the syntheses of carbasugars. By using methods of metabolic-pathway engineering, the Escherichia coli genes entB and entC, which encode isochorismatase and isochorismate synthase, were cloned and over-expressed in E. coli strains with a deficiency of entA, which encodes 2,3-dihydroxybenzoate synthase. A 30-fold increase in the corresponding EntB/EntC enzyme activities affects the accumulation of 2,3-trans-CHD in the cultivation medium. Although the strains did not contain deletions in chorismate-utilising pathways towards aromatic amino acids, neither chorismate nor any other metabolic intermediates were found as by-products. Fermentation of these strains in a 30 L pH-controlled stirred tank reactor showed that 2,3-trans-CHD could be obtained in concentrations of up to 4.6 g L(-1). This demonstrates that post-chorismate metabolites are accessible on a preparative scale by using techniques of metabolic-pathway engineering. Isolation and separation from fermentation salts could be performed economically in one step through anion-exchange chromatography or, alternatively, by reactive extraction. Starting from 2,3-trans-CHD as an example, we established short syntheses towards new carbasugar derivatives.

Benzoates↗
PubMed2003

One-pot synthesis of L-Fructose using coupled multienzyme systems based on rhamnulose-1-phosphate aldolase.

Two methods have been developed for the highly efficient enzymatic synthesis of L-fructose: one is based on rhamnulose-1-phosphate aldolase and acid phosphatase using racemic glyceraldehyde and dihydroxyacetone phosphate as substrates; the other is to generate enantiomerically pure L-glyceraldehyde in situ from glycerol for the aldol reaction, using galactose oxidase catalyzed oxidation of glycerol in the presence of catalase. Using this four-enzyme system, enantiomerically pure L-fructose was obtained. Using the more expensive dihydroxyacetone phosphate, the yield was 55% after purification.

Acid Phosphatase↗
PubMed2003

Directed evolution of N-acetylneuraminic acid aldolase to catalyze enantiomeric aldol reactions.

Expanding the scope of substrate specificity and stereoselectivity is of current interest in enzyme catalysis. Using error-prone PCR for in vitro directed evolution, the Neu5Ac aldolase from Escherichia coli has been altered to improve its catalytic activity toward enantiomeric substrates including N-acetyl-L-mannosamine and L-arabinose to produce L-sialic acid and L-KDO, the mirror-image sugars of the corresponding naturally occurring D-sugars. The first generation variant containing two mutations (Tyr98His and Phe115Leu) outside the (alpha,beta)(8)-barrel active site exhibits an inversion of enantioselectivity toward KDO and the second generation variant contains an additional amino acid change Val251Ile outside the alpha,beta-barrel active site that improves the enantiomeric formation of L-sialic acid and L-KDO. The X-ray structure of the triple mutant epNanA.2.5 at 2.3A resolution showed no significant difference between the wild-type and the mutant enzymes. We probed the potential structural 'hot spot' of enantioselectivity with saturation mutagenesis at Val251, the mutated residue most proximal to the Schiff base forming Lys165. The selected variant had an increase in k(cat) via replacement with another hydrophobic residue, leucine. Further sampling of a larger sequence space with error-prone PCR selected a third generation variant with significant improvement in L-KDO catalysis and a complete reversal of enantioselectivity.

Aldehydes↗
PubMed2001

Synthesis of Functionalized Cyclohexadiene-trans-Diols with Recombinant Cells of Escherichia coli D.F. was financially supported by the Graduiertenförderung des Landes NRW and the Studienstiftung des deutschen Volkes. We thank Professor Dr. Klaus Hantke, Universität Tübingen, for kindly providing the E. coli mutants H1882 and AN193. We thank Professor Dr. Eckhard Leistner, Universität Bonn, and Dr. Rolf Müller, GBF Braunschweig, for helpful discussions. Skillful technical assistance of Mrs. Ursula Degner and Ms. Regine Halbach is gratefully acknowledged. We thank Professor Dr. Christian Wandrey for his continuous and generous support.

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Journal Article↗