Enantioselective Formation and Ring-Opening of Epoxides Catalysed by Halohydrin Dehalogenases

View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UniversityBiocatalysis of Groningen Digital 291 Archive Enantioselective formation and ring-opening of epoxides catalysed by halohydrin dehalogenases D.B. Janssen*1, M. Majeric-Elenkov*´ 2, G. Hasnaoui*, B. Hauer† and J.H. Lutje Spelberg*3 *Biochemical Laboratory, Groningen Biomolecular Sciences and Biotechnology Institute, Groningen, The Netherlands, and †BASF AG, Fine Chemicals Research, Ludwigshafen, Germany Abstract Halohydrin dehalogenases catalyse the conversion of vicinal halohydrins into their corresponding epoxides, while releasing halide ions. They can be found in several bacteria that use halogenated alcohols or compounds that are degraded via halohydrins as a carbon source for growth. Biochemical and structural studies have shown that halohydrin dehalogenases are evolutionarily and mechanistically related to enzymes of the SDR (short-chain dehydrogenase/reductase) superfamily. In the reverse reaction, which is epoxide-ring opening, different nucleophiles can be accepted, including azide, nitrite and cyanide. This remarkable catalytic promiscuity allows the enzymatic production of a broad range of β-substituted alcohols from epoxides. In these oxirane-ring-opening reactions, the halohydrin dehalogenase from Agrobacterium radiobacter displays high enantioselectivity, making it possible to use the enzyme for the preparation of enantiopure building blocks for ﬁne chemicals. Introduction and stopped flow fluorescence measurements have indicated The bacterial degradation of epichlorohydrin, 2,3-dibromo- that the release of the halide ion from the active site is the 1-propanol, 1,2-dibromoethane and some related compounds slowest step in the catalytic cycle [4]. Based on phylogenetic involves a step in which a vicinal halohydrin is converted relationships, the known halohydrin dehalogenases can be into the corresponding epoxide and free halide. This intra- classified into group A, group B or group C (HheC) enzymes molecular substitution reaction is catalysed by halohydrin [2]. Only the structure of HheC from Agrobacterium radio- dehalogenases. Several different halohydrin dehalogenase bacter has been published, but the sequences suggest that genes have been cloned and expressed in Escherichia coli the epoxide-binding sites and halide-binding sites seem to [1,2]. Sequence analysis, mutation studies and the X-ray be rather different, which is in agreement with variations in structure of the Agrobacterium radiobacter HheC enzyme enantioselectivity and substrate spectrum. showed that these proteins belong to the SDR (short- In addition to mechanistic and kinetic investigations, re- chain dehydrogenase/reductase) superfamily of enzymes. cent work has been aimed at expanding the biocatalytic The HheC protein and other halohydrin dehalogenases are potential of these dehalogenases. It was found in 1968 by tetrameric enzymes with subunits of 27 kDa in which the Castro and Bartnicki [5] that a halohydrin dehalogenase, topology and catalytic groups are conserved, but they do not then termed halohydrin epoxidase, could catalyse a transhalo- have a binding site for a nicotinamide co-factor. Both the genation reaction. Later on, the enzymes were used for the halohydrin dehalogenases and the SDR enzymes possess a non-enantioselective exchange of a halogen substituent with conserved catalytic tetrad that is involved in abstraction of a nitrile group [6]. More recent studies have widened the a proton from the alcohol substrate and proton export from range of epoxide substrates that can be converted and have the active site. This tetrad is composed of Ser/Tyr/Arg/Asp identified new nucleophiles that can be used in the reverse in halohydrin dehalogenases and of Ser/Tyr/Lys/Asp in SDR reaction, allowing enantioselective nucleophilic epoxide ring enzymes (Figure 1A). In the case of SDR proteins, the opening. negative charge of the deprotonated hydroxy group oxygen is transferred via a hydride to the NAD(P)+ co-factor and a carbonyl group is formed. In halohydrin dehalogenases, the Biocatalysis with halohydrin developing oxyanion attacks the neighbouring carbon atom dehalogenases to which the halogen is bound, resulting in a nucleophilic displacement of the halogen and formation of an epoxide Enantioselective ring-closure of halohydrins [2,3]. The HheC protein has a distinct halide-binding site, The enantioselectivity that halohydrin dehalogenases display when catalysing ring-closure reactions makes them promising Key words: azide, biocatalysis, cyanide, epoxide, halohydrin dehalogenase, nitrite. biocatalysts for the production of optically active epoxides Abbreviations used: e.e., enantiomeric excess; SDR, short-chain dehydrogenase/reductase. and β-substituted alcohols by kinetic resolution. Whole- 1 To whom correspondence should be addressed (email [email protected]). 2 Present address: Rudjer Boskovic Institute, Bijenicka c. 54, 10000 Zagreb, Croatia. cell processes were developed by Kasai and co-workers for 3 Present address: Enzis, Nijenborgh 4, 9747 AG Groningen, The Netherlands. the production of optically active 2,3-dichloropropanol and C 2006 Biochemical Society 292 Biochemical Society Transactions (2006) Volume 34, part 2 Figure 1 Active site of halohydrin dehalogenase from A. radiobacter AD1 (A) Catalytic mechanism with deprotonation of the substrate hydroxy group by Tyr145, assisted by Ser132 and Arg149. (B) Schematic view of the unproductive binding of the non-converted (S)-enantiomer of p-nitrostyrene oxide in the active site [10]. 1-chloro-2,3-propanediol, and these compounds served as a in the order Cl− > Br− > I−. In conversions that, owing to an building block for a variety of optically active compounds unfavourable thermodynamic equilibrium, do not proceed [7,8]. Although the enzymes that are responsible for the to completion, or in case of the use of an enzyme with a enantioselective degradation of halopropanols in these modest enantioselectivity, the enantiomeric excess (e.e.) or organisms were not characterized in much detail, it is likely the yield of the remaining enantiomer will be low, hinder- that halohydrin dehalogenases play a key role, at least in 1- ing the application of the enzyme in a kinetic resolution chloro-2,3-propanediol degradation. and reducing the product e.e. as conversion proceeds [11]. The halohydrin dehalogenase from A. radiobacter AD1 A way to overcome the reversibility problem is the use (HheC) can catalyse the enantioselective conversion of var- of a tandem reaction. In this conversion, the epoxide that ious aromatic halohydrins, yielding enantiomerically en- is produced by HheC from A. radiobacter was trapped riched (R)-epoxides and (S)-halohydrins [9]. A structural away by an epoxide hydrolase originating from the same explanation for the high enantioselectivity of the enzyme was organism, thereby producing the diol [9]. Using this system, recently obtained by Dijkstra’s group by X-ray crystallo- reactions were drawn to completion, and the remaining (S)- graphic studies, using the two enantiomers of p-nitrostyrene enantiomers of the substrates 2,3-dichloro-1-propanol and oxide [10]. It appeared that the non-converted (S)-enantiomer 2-chloro-1-phenylethanol could be obtained at >99% e.e., binds in a non-productive conformation with the epoxide with apparent E values of >100 and 73 respectively. oxygen not oriented toward the Tyr-Ser pair, but in the Mutants of HheC with a higher rate of conversion of opposite direction, where it makes contact with a water mol- (R)-1-(p-nitrophenyl)-2-bromoethanol and 1,3-dichloro-2- ecule in the halide-binding site (Figure 1B). In this way, the propanol into their corresponding epoxides were obtained p-nitrophenyl group of the two enantiomers of the substrate by site-directed mutagenesis of the halide-binding site [12]. can be bound in a similar position, but the epoxide ring- This site is partially formed by a short loop that is stabilized opening reaction cannot occur for the (S)-enantiomer because by hydrogen bonds that connect the phenolic hydroxy group the epoxide oxygen is disoriented. Free-energy calculations of Tyr187 with the Oδ1atomofAsn176 and Nε1atomofTrp249. obtained by molecular dynamics simulations showed that the Both the Y187F and the W249F mutants showed an enhanced difference in binding energy between the two enantiomers kcat and an increased rate of halide release, as measured by is rather small. The difference corresponds to the loss of a stopped flow fluorescence experiments. The fact that a mutant single hydrogen bond in the enzyme complexed with the non- with a higher kcat showed faster halide release is in agree- preferred (S)-enantiomer, which is in agreement with the pre- ment with the earlier proposal that halide release is the rate- sence of two hydrogen bonds between the epoxide oxygen limiting step in the conversion of these substrates [4]. and enzyme in the case of the (R)-enantiomer (with serine and The E value in halohydrin dehalogenation reactions tyrosine), and only a single hydrogen-bond (with water) could also be improved by site-directed mutagenesis. The for the (S)-enantiomer. aformentioned W249F mutant showed an enhanced enantio- Whether the ring-closure reactions catalysed by halo- selectivity of 1-(p-nitrophenyl)-2-bromoethanol dehalogen- hydrin dehalogenases go to completion depends, among ation as compared with the wild-type [12]. The affected other things, on the type of (pseudo)halogen substituent. Trp 249 is coming

Load more