Arylmalonate decarboxylase (AMDase; EC 184.108.40.206) isolated from Alcaligenes bronchisepticus
strain KU1201 catalyses the enantioselective decarboxylation of prochiral α-aryl-α-methylmalonates to optically active α-arylpropionates with a high enantiomeric excess in high yield (Fig. 1
; Miyamoto & Ohta, 1990
; Ohta, 1997
). Unlike other decarboxylases, the enzyme requires no additional cofactors such as metal ions, coenzyme A or ATP (Miyamoto & Ohta, 1992a
). AMDase, comprising 240 amino-acid residues (molecular weight 24 737 Da), shows low homology to racemases and isomerases such as glutamate racemases (GluRs; Gallo & Knowles, 1993
; Hwang et al.
), aspartate racemase (AspR; Yohda et al.
; Liu et al.
) and other racemase-related enzymes such as hydanotine racemase (Watabe et al.
) and maleate cis
isomerase (Hatakeyama et al.
). GluR and AspR have been classified into the pyridoxal-5′-phosphate-independent racemase family (Tanner, 2002
; Gerlt et al.
). An alignment of the primary sequences of family members (Fig. 1
) suggests that Cys188 of AMDase is one of the essential residues forming the active site. This residue is thought to act as a proton donor to the intermediate, the enolate form of α-arylpropionate, from only one side of the enantiomeric face to produce the (R
)-product (Matoishi et al.
; Terao et al.
). AMDase becomes inactive on Cys188Ser mutation (Miyazaki et al.
) or in the presence of SH reagents such as iodoacetate, p
(Miyamoto & Ohta, 1992b
), suggesting a crucial role of the cysteine residue in the reaction.
Figure 1 (a) Scheme showing the enantioselective decarboxylation reaction of phenylmalonate catalyzed by AMDase. (b) An alignment of the amino-acid sequences of AMDase, GluR (Gallo & Knowles, 1993 ), AspR (Yohda et al., 1991 (more ...)
In the crystal structures of GluR from Aquifex pyrophilus
(Hwang et al.
) and AspR from Pyrococcus horikoshii
(Liu et al.
), two cysteine residues are symmetrically arranged in the active-site cleft and provide a reaction field that enables a two-base mechanism for their racemization reaction (Tanner, 2002
; Gallo et al.
; Glavas & Tanner, 1999
). In AMDase, Cys188 corresponds to one of the cysteine residues of the racemases and Gly74 takes the place of the other. This difference probably causes an asymmetric structure of the active site, enabling the enantioselective reaction. In fact, a Gly74Cys mutation converts AMDase to an enzyme that catalyses the racemization of α-arylpropionate (Terao et al.
) and Gly74Cys/Cys188Ser-mutated AMDase produces arylpropionate of the opposite enantiomorph to that of the wild-type enzyme, although the enzymatic reaction proceeds with a slower rate than that of the wild type (Ijima et al.
; Miyamoto et al.
). Thus, AMDase is of particular interest with regard to this conversion of the function by simple mutations and the three-dimensional structure of AMDase is indispensable in order to understand the reaction mechanism and for the design and development of novel enzymes (Terao et al.
Here, we describe the crystallization of AMDase and preliminary X-ray diffraction experiments at cryogenic temperature. The X-ray diffraction data suggested that 4–6 AMDase molecules occupy the crystallographic asymmetric unit. In addition, the quaternary structure of AMDase was studied by small-angle X-ray scattering (SAXS) measurements in order to determine the functional unit in solution.