Expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic Rubrobacter xylanophilus.
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.
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary ; Institute of Enzymology, Research Centre for Natural Sciences of Hungarian Academy of Sciences, Budapest, Hungary.
Klaudia
K
Kovács
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary.
Gergely
G
Bánóczi
Biocatalysis Research Group, Babeş-Bolyai University of Cluj-Napoca, Cluj-Napoca, Romania.
Andrea
A
Varga
Biocatalysis Research Group, Babeş-Bolyai University of Cluj-Napoca, Cluj-Napoca, Romania.
Izabella
I
Szabó
Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Budapest, Hungary.
András
A
Holczinger
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary.
Gábor
G
Hornyánszky
Institute of Enzymology, Research Centre for Natural Sciences of Hungarian Academy of Sciences, Budapest, Hungary.
Imre
I
Zagyva
Biocatalysis Research Group, Babeş-Bolyai University of Cluj-Napoca, Cluj-Napoca, Romania.
Csaba
C
Paizs
Institute of Enzymology, Research Centre for Natural Sciences of Hungarian Academy of Sciences, Budapest, Hungary ; Department of Applied Biotechnology and Food Science, Budapest University of Technology and Economics, Budapest, Hungary.
Beáta G
BG
Vértessy
Department of Organic Chemistry and Technology, Budapest University of Technology and Economics, Budapest, Hungary.
László
L
Poppe
D039903:Actinobacteria / Q000201:enzymology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D017422:Sequence Analysis, DNA; D017385:Sequence Homology; D013045:Species Specificity
D017931:DNA Primers; D010650:Phenylalanine Ammonia-Lyase