Elsevier

Bioorganic Chemistry

Volume 34, Issue 6, December 2006, Pages 345-361
Bioorganic Chemistry

Characterization of benzaldehyde lyase from Pseudomonas fluorescens: A versatile enzyme for asymmetric C–C bond formation

https://doi.org/10.1016/j.bioorg.2006.09.002Get rights and content

Abstract

The thiamin-diphosphate-dependent enzyme benzaldehyde lyase is a very import catalyst for chemoenzymatic synthesis catalyzing the formation and cleavage of (R)-hydroxy ketones. We have studied the stability of the recombinant enzyme and some enzyme variants with respect to pH, temperature, buffer salt, cofactors and organic cosolvents. Stability of BAL in chemoenzymatic synthesis requires the addition of cofactors to the buffer. Reaction temperature should not exceed 37 °C. The enzyme is stable between pH 6 and 8, with pH 8 being the pH-optimum of both the lyase and the ligase reaction. Potassium phosphate and Tris were identified as optimal reaction buffers and the addition of 20 vol% DMSO is useful to enhance both the solubility of aromatic substrates and products and the stability of BAL. The initial broad product range of BAL-catalyzed reactions has been enlarged to include highly substituted hydroxybutyrophenones and aliphatic acyloins.

Graphical abstract

Benzaldehyde lyase (BAL) is a very useful biocatalyst to access chiral 2-hydroxyketones from aldehydes. Various biochemical data important for the application of BAL in chemoenzymatic synthesis are presented.

  1. Download : Download full-size image

Introduction

Benzaldehyde lyase (BAL, EC 4.1.2.38) has so far been found only in Pseudomonas fluorescens Biovar I. This strain is able to utilize lignin-like compounds such as benzoin and anisoin as a sole carbon source. The enzyme responsible for this catabolic activity is BAL which catalyzes the cleavage of the aromatic acyloins to aldehydes, which are further catabolized in the β-ketoadipate pathway. BAL has been first described by Gonzales and Vicuna [1]. These authors established the requirement for thiamin diphosphate (ThDP) and a divalent cation such as Mg2+. Later the coding gene (bzl) was cloned and its DNA sequence has been published [2]. Recently the 3D structure of BAL has been elucidated by some of us [3], and catalytically important residues have been determined [4]. Although BAL was first identified by its lyase activity, we have previously reported that it is also able to catalyze the reverse reaction [5], [6]. Subsequently the enzyme has been used to catalyze the synthesis of various aromatic and heteroaromatic 2-hydroxy ketones [7], [8], [9], [10]. In contrast to wild-type benzoylformate decarboxylase (wtBFD) [11], BAL accepts aromatic aldehydes substituted in the ortho-position as well. Only a few aromatic aldehydes, such as pyridine 3- and 4-carbaldehyde as well as sterically exceedingly demanding aldehydes resulted in either very low yields or in no benzoin condensation at all [5]. Moreover, in addition to acetaldehyde, mono- and dimethoxy acetaldehyde are good acceptor substrates for BAL, providing enantiopure hydroxypropiophenone derivatives [12]. Various ThDP-dependent enzymes, especially 2-keto acid decarboxylases such as pyruvate decarboxylase (PDC) [13] and BFD [11], [14], [15], have been described to catalyze C–C bond formation and/or cleavage. Of the enzymes of this type, BAL possesses a remarkably broad substrate and reaction range [16], [17], [18], [19], making a vast variety of chiral 2-hydroxy ketones accessible [6], [20], [21]. In addition, since BAL is strictly (R)-enantioselective, the enzyme is also useful for kinetic resolution of racemic benzoin [5]. Some typical reactions catalyzed by BAL are shown in Fig. 1.

BAL catalyzes the cleavage and formation of (R)-2-hydroxyketones such as benzoin and 2-hydroxypropiophenone via a common intermediate ThDP-bound carbanion-enamine. Conversely, wild-type (wt) BAL does not show any decarboxylase activity towards 2-keto acids. However, this activity can be introduced by a single point mutation (BALA28S [4]). The enantioselectivity and the substrate specificity of BAL have recently been explained by molecular modelling based on the 3D structure of the enzyme [22].

We have re-cloned the BAL gene (blz) [2] in order to obtain an efficient expression system for BAL and have corrected some errors in the original DNA sequence [23]. The recombinant enzyme carrying a C-terminal hexahistidine tag was thoroughly characterized with respect to its carboligase and carbolyase activity, its stability, pH- and temperature optima, kinetic parameters, native molecular weight, and isoelectric point. Mutagenesis studies based on sequence and structural comparison with BFD from Pseudomonas putida revealed A480 in BAL as a key residue in the carboligase and carbolyase reactions. Further, we describe the BAL catalyzed synthesis of hydroxybutyrophenones, which opens up a new chemoenzymatic access to chiral polyols.

Section snippets

Re-cloning of BAL

PCR amplification of the BAL gene (blz) (1.7 kb) was performed using the plasmid pUC18/blz [2] as a template and the following primers, which introduce appropriate restriction sites for the subsequent cloning steps:

BAL-s5′-CCA TGGCGA TGA TTA CAG GCG GCGAA-3′
NcoI
BAL-as5′-GGA TCC GAA GGG GTC CAT GCC GAT CAG AA-3′
BamHI

The BAL gene was first ligated into the vector pUC18, which was linearized with SmaI and dephosphorylated prior to ligation, in order to allow digestion with NcoI. The resulting

Cloning and expression

In order to improve the expression yield and to simplify purification of the enzyme a vector, pBALhis, was prepared. This allows the expression of BAL with a C-terminal His-tag upon induction with 1-isopropyl-β-thiogalactoside (IPTG). Sequencing of the BAL gene in pBALhis yielded significant differences to the published sequence. Overall, three insertions, three deletions and two base exchanges were found in the range of nucleotides 210–299. These resulted in changes to the identity of 29 amino

Conclusions

In this study we have investigated optimal reaction conditions for BAL in more detail.

The application of BAL in chemoenzymatic synthesis requires the addition of cofactors to the buffer: 2.5 mM MgSO4 and 0.1 mM ThDP are sufficient to keep the enzyme stable at up to maximal 37 °C (Fig. 3). The enzyme is stable between pH 6 and 8, with pH 8 being the pH-optima of the lyase and ligase reaction (Fig. 6). Optimal buffers are potassium phosphate and Tris (Fig. 5).

As water-miscible organic cosolvents

Acknowledgments

E. Janzen is grateful to “Deutsche Graduiertenförderung der Konrad-Adenauer Stiftung” for financial support. We are thankful to Ralf Feldmann for skilful technical assistance. Financial support by the DFG in the scope of SFB 380 (M. Pohl, M. Müller), and from an NSF FIBR project grant (M. McLeish) is gratefully acknowledged.

References (32)

  • P. Hinrichsen et al.

    Gene

    (1994)
  • M.M. Kneen et al.

    BBA-Proteins Proteom.

    (2005)
  • N. Kurlemann et al.

    Tetrahedron: Asymmetry

    (2004)
  • A.S. Demir et al.

    Tetrahedron

    (2004)
  • T. Hischer et al.

    Tetrahedron

    (2005)
  • U. Schörken et al.

    BBA

    (1998)
  • A.S. Demir et al.

    Tetrahedron: Asymmetry

    (1999)
  • D.J. Korz et al.

    J. Biotechnol.

    (1995)
  • M.M. Bradford

    Anal. Biochem.

    (1976)
  • B. Gonzalez et al.

    J. Bacteriol.

    (1989)
  • T.G. Mosbacher et al.

    FEBS J.

    (2005)
  • A.S. Demir et al.

    J. Chem. Soc. Perkin Trans.

    (2001)
  • P. Dünkelmann et al.

    J. Am. Chem. Soc.

    (2002)
  • M. Sanchez-Gonzalez et al.

    Adv. Syn. Cat.

    (2003)
  • H. Iding et al.

    Chem. Eur. J.

    (2000)
  • A.S. Demir et al.

    Org. Lett.

    (2003)
  • Cited by (0)

    View full text