Expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic Rubrobacter xylanophilus

Klaudia Kovaćs, Gergely Bańóczi, Andrea Varga, Izabella Szabó, András Holczinger, Gábor Hornyánszky, Imre Zagyva, Csaba Paizs, B. Vértessy, L. Poppe

Research output: Contribution to journalArticle

16 Citations (Scopus)

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.

Original languageEnglish
Article numbere85943
JournalPLoS One
Volume9
Issue number1
DOIs
Publication statusPublished - Jan 27 2014

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Rubrobacter xylanophilus
Phenylalanine Ammonia-Lyase
His-His-His-His-His-His
phenylalanine ammonia-lyase
Bacteria
circular dichroism spectroscopy
Affinity chromatography
Circular Dichroism
Gene encoding
Enzymes
Phenylalanine
Recombinant Proteins
Disulfides
Escherichia coli
Electrostatics
Assays
Screening
Salts
Genes
Amino Acids

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic Rubrobacter xylanophilus. / Kovaćs, Klaudia; Bańóczi, Gergely; Varga, Andrea; Szabó, Izabella; Holczinger, András; Hornyánszky, Gábor; Zagyva, Imre; Paizs, Csaba; Vértessy, B.; Poppe, L.

In: PLoS One, Vol. 9, No. 1, e85943, 27.01.2014.

Research output: Contribution to journalArticle

Kovaćs, K, Bańóczi, G, Varga, A, Szabó, I, Holczinger, A, Hornyánszky, G, Zagyva, I, Paizs, C, Vértessy, B & Poppe, L 2014, 'Expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic Rubrobacter xylanophilus', PLoS One, vol. 9, no. 1, e85943. https://doi.org/10.1371/journal.pone.0085943
Kovaćs, Klaudia ; Bańóczi, Gergely ; Varga, Andrea ; Szabó, Izabella ; Holczinger, András ; Hornyánszky, Gábor ; Zagyva, Imre ; Paizs, Csaba ; Vértessy, B. ; Poppe, L. / Expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic Rubrobacter xylanophilus. In: PLoS One. 2014 ; Vol. 9, No. 1.
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