Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus

• DOI: 10.1371/journal.pone.0085943
• Publication Date: 2014-01-27T21:25:00Z
• Authors: Klaudia Kovács; Gergely Bánóczi; Andrea Varga; Izabella Szabó; András Holczinger; Gábor Hornyánszky; Imre Zagyva; Csaba Paizs; Beáta G. Vértessy; László Poppe
• Keywords: Models, Molecular; 0301 basic medicine; Protein Conformation; Science; Molecular Sequence Data; Sequence Homology; Chromatography, Affinity; 03 medical and health sciences; Species Specificity; Escherichia coli; Amino Acid Sequence; Cloning, Molecular; QH301 Biology / biológia; DNA Primers; Phenylalanine Ammonia-Lyase; Base Sequence; Circular Dichroism; Q; R; Sequence Analysis, DNA; Hydrogen-Ion Concentration; 3. Good health; Actinobacteria; QD04 Organic chemistry / szerves kémia; Medicine; Research Article

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 α-helical 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.