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

Abstract

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.

Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae

“…The high pH optimum of the reactions may be a reflection of the reaction mechanism and is generally identical for all enzymes and similar to what has been observed previously for RsTAL, SeSam8, and other homologous enzymes (18,20,21,23,27,(78)(79)(80)(81). Alkaline pH may also be used for biocatalysis, where tyrosine produced by fermentation at physiological pH can be converted to pHCA in high titers by subsequent reaction at alkaline pH in the presence of TAL-expressing E. coli cells or cell paste (82).…”

From Synthetic Chemistry and Stereoselective Biotransformations to Enzyme Biochemistry – The Bioorganic Chemistry Group at the Budapest University of Technology and Economics

“…The extremely high pH optimum of RxPAL could be rationalized by a homology model indicating possible disulfide bridges (Fig. 11D), extensive salt-bridge formation and an excess of negative electrostatic potential on the surface [66]. Histidine, phenylalanine and tyrosine ammonia-lyases (HAL, PAL and TAL) all catalyze ammonia elimination with the aid of a post-translationally formed electrophilic prosthetic group (MIO) [25][26][27].…”

“…The sequence of a PAL of the thermophilic bacterium Rubrobacter xylanophilus (RxPAL) was identified by screening the genomes of bacteria for members of the PAL family and its gene was cloned and overexpressed in E. coli [66]. The extremely high pH optimum of RxPAL could be rationalized by a homology model indicating possible disulfide bridges (Fig.…”

Aminated Single-walled Carbon Nanotubes as Carrier for Covalent Immobilization of Phenylalanine Ammonia-lyase

“…As it was known that ammonia addition onto arylacrylates can be performed at lower ammonia concentrations [19,29], the B A reaction was tested with 2 in 3 M and 2 M ammonia solutions as well. At the lower ammonia concentrations SwCNT NH2 -PAL exhibited significantly higher stability (Fig.…”