Eduardo García-Junceda

Enzyme catalysed tandem reactions

To transfer to the laboratory, the excellent efficiency shown by enzymes in Nature, biocatalysis, had to mimic several synthetic strategies used by the living organisms. Biosynthetic pathways are examples of tandem catalysis and may be assimilated in the biocatalysis field for the use of isolated multi-enzyme systems in the homogeneous phase. The concurrent action of several enzymes that work sequentially presents extraordinary advantages from the synthetic point of view, since it permits a reversible process to become irreversible, to shift the equilibrium reaction in such a way that enantiopure compounds can be obtained from prochiral or racemic substrates, reduce or eliminate problems due to product inhibition or prevent the shortage of substrates by dilution or degradation in the bulk media, etc. In this review we want to illustrate the developments of recent studies involving in vitro multi-enzyme reactions for the synthesis of different classes of organic compounds.

Multi-Step Enzyme Catalysis: Biotransformations and Chemoenzymatic Synthesis

Multi-Enzyme Synthesis of Glycoconjugates Multimodular Synthases as Tools of the Synthetic Chemist Modifying the Glycosilation Pattern in Microorganisms by Combinatorial Biosynthesis Chemoenzymatic Routes to Enantiomerically Pure Amines and Aminoacids Asymmetric Transformations by Couples Enzymes and Metal Catalysis Engineering Novel Biosynthetic Pathways to Produce New Chemical Compounds Biocatalysis and Chemical Catalysis for the Synthesis of Intermediates of Pharmaceutical Interest Oxidizing Enzymes into Multi-Enzyme Biotransformation Processes The Role of Host-Intrinsic Enzyme Activities in Recombinant Whole-Cells Catalyzed Processes Microbial Production of DNA Building Blocks Enzyme Initiation of Asymmetric Cascade-Reactions The System of Biotransformations Approach for Multi-Step Processes Chemoenzymatic Synthesis of Homogeneous Glycoprotein Aldolases Catalyzed C-C Bond Formation in the Core of Multi-Step Processes

From kinase to cyclase: an unusual example of catalytic promiscuity modulated by metal switching.

“This is the pre-peer reviewed version of the following article: Sanchez-Moreno, I., Iturrate, L., Martin-Hoyos, R., Jimeno, M. L., Mena, M., Bastida, A. and Garcia-Junceda, E. (2009) From Kinase to Cyclase: An Unusual Example of Catalytic Promiscuity Modulated by Metal Switching. ChemBioChem. 10, 225-229, which has been published in final form at http://www3.interscience.wiley.com/journal/121544668/abstract?CRETRY=1&SRETRY=0.”

Preparation and characterization of a bifunctional aldolase/kinase enzyme. A more efficient biocatalyst for C-C bond formation

A bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening linker of five amino acid residues. The fusion protein was expressed soluble and retained both kinase and aldolase activities. The secondary structures of the bifunctional enzyme and the parental enzymes were analyzed by circular dichroism (CD) spectroscopy to study the effect of the covalent coupling of the two parent proteins on the structure of the fused enzyme. Because S. carnosus FBPA is a thermostable protein, the effect of the fusion on the thermal stability of the bifunctional enzyme has also been studied. The proximity of the active centers in the fused enzyme promotes a kinetic advantage as the 20-fold increment in the initial velocity of the overall aldol reaction indicates. Experimental evidence supports that this increase in the reaction rate can be explained in terms of substrate channeling.

Substrate channelling in an engineered bifunctional aldolase/kinase enzyme confers catalytic advantage for C–C bond formation

A new bifunctional enzyme that displays both aldolase and kinase activities has been designed and successfully used in the synthesis of aldol adducts, employing DHA as initial donor, with an increase in the reaction rate of 20-fold over the parent enzymes, which can be interpreted in terms of substrate channelling.

Synthesis of Carba- and C-Fucopyranosides and Their Evaluation as α-Fucosidase Inhibitors − Analysis of an Unusual Conformation Adopted by an Amino-C-fucopyranoside

Several carba- and C-fucopyranosides bearing a variety of substituents at their pseudoanomeric positions have been synthesized and tested as inhibitors of bovine kidney α-fucosidase. From the IC50 values, some conclusions about the structure-activity relationship could be drawn. The presence of a hydroxyl group close to the pseudoanomeric position, or the presence of an aromatic ring as substituent, were beneficial for inhibitory activity. A 1-(R)-amino-phenylmethyl C-fucopyranoside, the activity of which increased 40 times when the pH was changed from 5.0 to 7.0, adopted a 4C1 conformation, in contrast to the 1C4 form normally adopted by natural fucopyranosides. The conformation of this compound was analyzed in comparison with that of the 1-(S)-configured epimer, using NMR spectroscopy and molecular modeling.

In Vivo Chaperone‐Assisted Folding of α‐1,6‐Fucosyltransferase from Rhizobium sp.

3 pages, 3 figures.-- PMID: 12794864 [PubMed].-- Supplementary information (Materials and methods, 8 pages) available at: http://www.wiley-vch.de/contents/jc_2268/2003/z514_s.pdf

Tuning the Phosphoryl Donor Specificity of Dihydroxyacetone Kinase from ATP to Inorganic Polyphosphate. An Insight from Computational Studies

Dihydroxyacetone (DHA) kinase from Citrobacter freundii provides an easy entry for the preparation of DHA phosphate; a very important C3 building block in nature. To modify the phosphoryl donor specificity of this enzyme from ATP to inorganic polyphosphate (poly-P); a directed evolution program has been initiated. In the first cycle of evolution, the native enzyme was subjected to one round of error-prone PCR (EP-PCR) followed directly (without selection) by a round of DNA shuffling. Although the wild-type DHAK did not show activity with poly-P, after screening, sixteen mutant clones showed an activity with poly-phosphate as phosphoryl donor statistically significant. The most active mutant presented a single mutation (Glu526Lys) located in a flexible loop near of the active center. Interestingly, our theoretical studies, based on molecular dynamics simulations and hybrid Quantum Mechanics/Molecular Mechanics (QM/MM) optimizations, suggest that this mutation has an effect on the binding of the poly-P favoring a more adequate position in the active center for the reaction to take place.

Aryl Sulfotransferase from Haliangium ochraceum: A Versatile Tool for the Sulfation of Small Molecules

Sulfation is an important molecular modification that regulates essential cellular processes and is also implicated in numerous pathological processes. The enzymes responsible for this reaction in living organisms are sulfotransferases. The gene Hoch_5094 from Haliangium ochraceum is annotated as a putative sulfotransferase. The arylsulfotransferase codified by this gene (HocAST) was expressed heterologously in E. coli and showed aryl sulfotransferase activity. Circular dichroism analysis of HocAST showed a main α/β secondary structure that agrees with the overall structure of other cytosolic sulfotransferases. Interestingly, HocAST was able to use both p‐nitrophenyl sulfate and 3′‐phosphoadenosine‐5′‐phosphosulfate (PAPS) as sulfuryl donors contrary to that of aryl sulfate sulfotransferase, which cannot use PAPS as a donor. Regarding the specificity towards the acceptor, HocAST has shown quite a wide scope and was able to accept several mono‐ and dihydroxylated phenols and other phosphorylated compounds as substrates.

Enzymatic synthesis of disaccharides by β-galactosidase-catalyzed glycosylation of a glycocluster

The synthesis of disaccharides by galactosylation of a glucose-containing cluster has been studied using β-galactosidases from different sources. Galactosylation of the cluster could be detected by ESI-MS. After size-exclusion chromatography of the reaction mixtures and subsequent cleavage of the linker, galactopyranosyl-glucose disaccharides were obtained with yield and regioselectivity dependent on the origin of the enzyme. The possibility of using this method for the synthesis of multivalent glycoconjugates is discussed.