The sequence of a phenylalanine ammonia-lyase (PAL; EC: 4.3.1.24) of the thermophilic and radiotolerant bacterium
Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the phenylalanine
ammonia-lyase family. A synthetic gene encoding the RxPAL protein was cloned and overexpressed in Escherichia coli TOP
10 in a soluble form with an N-terminal His6-tag and the recombinant RxPAL protein was purified by Ni-NTA affinity
chromatography. The activity assay of RxPAL with L-phenylalanine at various pH values exhibited a local maximum at pH 8.5
and a global maximum at pH 11.5. Circular dichroism (CD) studies showed that RxPAL is associated with an extensive ahelical
character (far UV CD) and two distinctive near-UV CD peaks. These structural characteristics were well preserved up
to pH 11.0. The extremely high pH optimum of RxPAL can be rationalized by a three-dimensional homology model
indicating possible disulfide bridges, extensive salt-bridge formation and an excess of negative electrostatic potential on the
surface. Due to these properties, RxPAL may be a candidate as biocatalyst in synthetic biotransformations leading to
unnatural L- or D-amino acids or as therapeutic enzyme in treatment of phenylketonuria or leukemia
