Journal of Molecular Catalysis B: Enzymatic 61 (2009) 73–79 Contents lists available at ScienceDirect Journal of Molecular Catalysis B: Enzymatic journal homepage: www.elsevier.com/locate/molcatb Investigation of the carboligase activity of thiamine diphosphate-dependent enzymes using kinetic modeling and NMR spectroscopy Mariya Kokova a , Michael Zavrel b , Kai Tittmann c , Antje C. Spiess b , Martina Pohl a,∗ a Institute of Molecular Enzyme Technology, Heinrich-Heine University Düsseldorf, 52426 Jülich, Germany b AVT–Biochemical Engineering, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany c Albrecht-von-Haller-Institut, Georg-August-Universität Göttingen, Justus-von-Liebig-Weg 11, D-37077 Göttingen, Germany article info Article history: Available online 9 March 2009 Keywords: Thiamine diphosphate-dependent enzymes Kinetic modeling NMR spectroscopy Carboligation Reaction mechanism Intermediates abstract The benzoin condensation reaction catalyzed by the thiamine diphosphate (ThDP)-dependent enzymes benzaldehyde lyase (BAL) and benzoylformate decarboxylase variant His281Ala (BFDH281A) was studied via initial rate measurements, progress curve analysis and NMR-based analysis of reaction intermediates. Using a mechanistic kinetic model, the kinetic parameters and microscopic rate constants were deter- mined, thus identifying the rate limiting steps of the reaction. In BAL, overall reaction is rate-limited by product release, whereas in BFDH281A substrate binding is the slowest step of catalysis. These results were further confirmed by analysis of covalent reaction intermediates using NMR spectroscopy after acid quench isolation. © 2009 Elsevier B.V. All rights reserved. 1. Introduction The benzoin condensation is a key C–C bond forming reaction between aromatic aldehydes, which is traditionally performed by chemical catalysts [1]. Alternatively, the reaction can be catalyzed by enzymes, especially when products of high enantiomeric purity are required. In that concern, the thiamine diphosphate (ThDP)- dependent enzymes benzaldehyde lyase and benzoylformate decarboxylase are successfully employed for chemo-enzymatic synthesis involving carboligation steps [2]. Benzaldehyde lyase (BAL, EC 4.1.2.38) from Pseudomonas fluo- rescens Biovar I has been first described by Gonzalez and Vicuna [3] as a thiamine diphosphate (ThDP)-dependent enzyme able to cleave the acyloin linkage of benzoin to yield two molecules of benzaldehyde. As the enzyme is strictly selective for conversion of the (R)-enantiomer, it can be applied in kinetic resolution of racemic benzoins [4,5]. Although BAL was initially described to show only lyase activity, it was later discovered that it can also cat- alyze the reverse reaction, that is the carboligation of benzaldehyde to (R)-benzoin with high enantiomeric excess (ee) > 99% [4,5]. More- over, the enzyme has been demonstrated to catalyze formation of various highly enantiopure (R)-hydroxypropiophenone derivatives, using short-chain aliphatic aldehydes as acceptors and aromatic ∗ Corresponding author at: Institute of Molecular Enzyme Technology, Heinrich- Heine Universität Düsseldorf, Research Center Jülich, 52426 Jülich, Germany. Tel.: +49 2461 613704; fax: +49 2461 612490. E-mail address: ma.pohl@fz-juelich.de (M. Pohl). aldehydes including ortho-substituted ones as donor substrates [6]. Benzoylformate decarboxylase (BFD) (EC 4.1.1.7) from Pseu- domonas putida was first reported by Wilcocks et al. [7]. The enzyme is involved in the non-oxidative decarboxylation of benzoylformate [8], and is moreover able to catalyze the enantioselective synthe- sis of (S)-2-hydroxypropanone derivatives as a side reaction [9]. BFD further catalyzes the ligation of a broad range of aromatic, heteroaromatic, conjugated olefinic aldehydes as donor substrates, preferably with acetaldehyde as an acceptor. Besides acetaldehyde, BFD converts aromatic and heteroaromatic substrates as acceptors to produce enantiopure (R)-benzoin and derivatives thereof, but in contrast to BAL with very low reaction rates [8]. The benzoin- forming activity of BFD was enhanced by site-directed mutagenesis of histidine 281 to alanine, yielding more space in the active site for accommodating larger acceptor aldehydes [9]. Therefore, the variant BFDH281A was chosen together with wild-type BAL for our research purposes. A severe technical problem is the low solubility of the benzoin substrates in aqueous buffer. Addition of 20–30% DMSO in the reac- tion mixture considerably improves the solubility of the substrates, and thus the enzymatic performance [10]. Both BAL and BFD are known to catalyze the benzoin formation from two benzaldehyde molecules enantioselectively (more than 99% ee) and high conver- sion (BAL 97% and BFD 70%, in the presence of DMSO) [9]. However, the effect of organic solvents on the stability and activity of these enzymes has not been characterized in detail. The catalytic cycle of the reaction catalyzed by BAL [4] can be subdivided into three main steps (Scheme 1). At first, the 1381-1177/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.molcatb.2009.02.021