Israel Sánchez-Moreno

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.

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.

7.20 Multi-Enzyme Reactions

To transfer to the laboratory, the excellent efficiency shown by enzymes in Nature, biocatalysis had to mimic several synthetic strategies used by Nature to achieve its objectives. Thus, the biosynthetic pathways developed by living beings may be assimilated in the biocatalysis field to the use of isolated multienzyme systems in the homogeneous phase. In this chapter, the authors have described and discussed several examples of the use of different multienzyme strategies in Asymmetric Synthesis.

Development of a new method for d-xylose detection and quantification in urine, based on the use of recombinant xylose dehydrogenase from Caulobacter crescentus.

The gene xylB from Caulobacter crescentus has been cloned and expressed in Escherichia coli providing a high yield of xylose dehydrogenase (XylB) production and excellent purity (97%). Purified recombinant XylB showed an absolute dependence on the cofactor NAD(+) and a strong preference for d-xylose against other assayed mono and disaccharides. Additionally, XylB showed strong stability when stored as freeze-dried powder at least 250days both at 4°C and room temperature. In addition, more than 80% of the initial activity of rehydrated freeze-dried enzyme remained after 150days of incubation at 4°C. Based on these characteristics, the capability of XylB in d-xylose detection and quantification was studied. The linearity of the method was maintained up to concentrations of d-xylose of 10mg/dL and the calculated limits of detection (LoD) and quantification (LoQ) of xylose in buffer were 0.568mg/dL and 1.89mg/dL respectively. Thus, enzymatic detection was found to be an excellent method for quantification of d-xylose in both buffer and urine samples. This method can easily be incorporated in a new test for the diagnosis of hypolactasia through the measurement of intestinal lactase activity.

Phosphorylation Catalyzed by Dihydroxyacetone Kinase

Siteand enantioselective kinases have been very useful catalysts for biocatalytic phosphorylations in straightforward syntheses of phosphorylated metabolites. Biocatalytic phosphorylations catalyzed by recombinant dihydroxyacetone-kinase beyond the dihydroxyacetone substrate have been investigated with quantitative P-NMR spectroscopy using pyruvate-kinase-catalyzed ATPregeneration. A nearly 100% conversion of D-glyceraldehyde to Dglyceraldehyde 3-phosphate has been found. Interestingly, with pure L-glyceraldehyde as substrate, practically no formation of Lglyceraldehyde 3-phosphate was observed. Catalytic asymmetric phosphorylations without the use of protecting groups for the phosphoryl group donors as well as for the substrates and the development of suitable catalysts are of major synthetic interest for the introduction of phosphoryl groups in a selective and sustainable way. As a large number of catalytic phosphorylation reactions play key roles in biological cells and are performed with excellent selectivity, it is no surprise that enzymes have also proven to be very suitable and versatile catalysts for asymmetric phosphorylation reactions from laboratory to industrial large-scale [1]. In contrast to chiral small molecular weight organocatalysts, where the two enantiomers can be used to catalyze the formation of the two enantiomeric products, the search for enantiocomplementary biocatalysts requires various different strategies [2]. For certain enzyme classes like hydrolases, alcohol dehydrogenases or transaminases both enantioselective enzymes have been found. Glycerol kinases have all led to products with the L-configuration and glycerol kinases with opposite enantioselectivity have so far not been discovered. Our aim was to extend the kinasecatalyzed asymmetric synthesis of L-glyceraldehyde 3phosphate (L-GAP) [3], which is toxic to cells, to the other enantiomer and major metabolite D-glyceraldehyde 3-phosphate (D-GAP). D-GAP was so far obtained by chemical synthesis or a one-pot enzymatic reaction sequence using the three enzymes fructose-bisphosphate aldolase, sn-glycerol 3-phosphate dehydrogenase and formate dehydrogenase [3]. In addition, in situ D-GAP synthesis has also been performed in different enzyme cascade reactions, such as acid phosphatase/DERA aldolases [4] and triosephosphate dehydrogenase/fructose-1,6biphosphate aldolase/FSA aldolase [5].Therefore we looked for suitable kinase candidates. Among the different kinases ATPdependent dihydroxyacetone kinases are of interest because they are able to differentiate between hydroxyand keto-groups. Dihydroxyacetone kinases (DHAK) are highly conserved and specific enzymes of microbial glycerol utilization pathways are occurring widely in plants, animals and some microorganisms. In an artificial pathway they catalyze the detoxification of dihydroxyacetone by phosphorylation [6] to the glycolytic intermediate dihydroxyacetone phosphate [7]. The roles of the active site residues of dihydroxyacetone kinases have been investigated by structural and mechanistic studies [8]. A number of dihydroxyacetone kinases have been applied in the straightforward preparation of dihydroxyacetone phosphate by catalyzing the phosphorylation of dihydroxyacetone (Figure 1) and using ATP as phosphoryl donor [9]. ATP regeneration in large scale reactions has been successfully solved by using two different cofactor recycling systems: (i) the enzyme acetate kinase (AK) with acetyl phosphate as final donor [10] and (ii) the enzyme pyruvate kinase and the substrate phosphoenol pyruvate [5]. Figure 1. Dihydroxyacetone kinase-catalyzed phosphorylations of dihydroxyacetone and D-glyceraldehyde using ATP as phosphoryl group donor and an enzymatic regeneration system for the ATP cofactor The dha operon expression is not only induced by dihydroxyacetone, but also by glyceraldehyde [11], which also fits into the active site of the ATP-dependent dihydroxyacetone kinase. As several ATP-dependent dihydroxyacetone kinases have been found to take racemic glyceraldehyde as substrate [12] and enantiomerically pure Dand L-glyceraldehyde became accessible [13], we investigated racemic glyceraldehyde as well as the enantiomerically pure Dand L-glyceraldehyde as substrates of the recombinant Citrobacter freundii dihydroxyacetone kinase (Figure 1), which was expressed in E. coli. Using a coupled enzymatic assay for measuring the DHAK activity (see experimental section) a value of 1.71 U/ml was obtained for the racemic glyceraldehyde, while with pure D[a] Dr. D.Gauss, Dr. R.Wohlgemuth (Corresponding Author) Sigma-Aldrich, Member of Merck Group Industriestrasse 25, CH-9470 Buchs, Switzerland E-mail: roland.wohlgemuth@sial.com [b] Dr. I.Sanchez-Moreno,[‡] Dr. I.Oroz-Guinea,[‡‡] Dr.E.Garcia-Junceda Departamento de Química Bioorgánica, Instituto de Química Orgánica General, CSIC (IQOG-CSIC), Madrid 28006, Spain [‡] Venter Pharma S.L. Azalea 1, 28109 Alcobendas (Madrid) [‡‡] Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis. Felix-Hausdorff-Str. 4. Greifswald D-17487 Germany

Analytical Validation of a New Enzymatic and Automatable Method for d-Xylose Measurement in Human Urine Samples

Hypolactasia, or intestinal lactase deficiency, affects more than half of the world population. Currently, xylose quantification in urine after gaxilose oral administration for the noninvasive diagnosis of hypolactasia is performed with the hand-operated nonautomatable phloroglucinol reaction. This work demonstrates that a new enzymatic xylose quantification method, based on the activity of xylose dehydrogenase from Caulobacter crescentus, represents an excellent alternative to the manual phloroglucinol reaction. The new method is automatable and facilitates the use of the gaxilose test for hypolactasia diagnosis in the clinical practice. The analytical validation of the new technique was performed in three different autoanalyzers, using buffer or urine samples spiked with different xylose concentrations. For the comparison between the phloroglucinol and the enzymatic assays, 224 urine samples of patients to whom the gaxilose test had been prescribed were assayed by both methods. A mean bias of −16.08 mg of xylose was observed when comparing the results obtained by both techniques. After adjusting the cut-off of the enzymatic method to 19.18 mg of xylose, the Kappa coefficient was found to be 0.9531, indicating an excellent level of agreement between both analytical procedures. This new assay represents the first automatable enzymatic technique validated for xylose quantification in urine.