Cargando…

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 wa...

Descripción completa

Detalles Bibliográficos
Autores principales: Kovács, Klaudia, Bánóczi, Gergely, Varga, Andrea, Szabó, Izabella, Holczinger, András, Hornyánszky, Gábor, Zagyva, Imre, Paizs, Csaba, Vértessy, Beáta G., Poppe, László
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903478/
https://www.ncbi.nlm.nih.gov/pubmed/24475062
http://dx.doi.org/10.1371/journal.pone.0085943
_version_ 1782301093440520192
author Kovács, Klaudia
Bánóczi, Gergely
Varga, Andrea
Szabó, Izabella
Holczinger, András
Hornyánszky, Gábor
Zagyva, Imre
Paizs, Csaba
Vértessy, Beáta G.
Poppe, László
author_facet Kovács, Klaudia
Bánóczi, Gergely
Varga, Andrea
Szabó, Izabella
Holczinger, András
Hornyánszky, Gábor
Zagyva, Imre
Paizs, Csaba
Vértessy, Beáta G.
Poppe, László
author_sort Kovács, Klaudia
collection PubMed
description 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 His(6)-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.
format Online
Article
Text
id pubmed-3903478
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher Public Library of Science
record_format MEDLINE/PubMed
spelling pubmed-39034782014-01-28 Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus Kovács, Klaudia Bánóczi, Gergely Varga, Andrea Szabó, Izabella Holczinger, András Hornyánszky, Gábor Zagyva, Imre Paizs, Csaba Vértessy, Beáta G. Poppe, László PLoS One 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 His(6)-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. Public Library of Science 2014-01-27 /pmc/articles/PMC3903478/ /pubmed/24475062 http://dx.doi.org/10.1371/journal.pone.0085943 Text en © 2014 Kovács et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Kovács, Klaudia
Bánóczi, Gergely
Varga, Andrea
Szabó, Izabella
Holczinger, András
Hornyánszky, Gábor
Zagyva, Imre
Paizs, Csaba
Vértessy, Beáta G.
Poppe, László
Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
title Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
title_full Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
title_fullStr Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
title_full_unstemmed Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
title_short Expression and Properties of the Highly Alkalophilic Phenylalanine Ammonia-Lyase of Thermophilic Rubrobacter xylanophilus
title_sort expression and properties of the highly alkalophilic phenylalanine ammonia-lyase of thermophilic rubrobacter xylanophilus
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3903478/
https://www.ncbi.nlm.nih.gov/pubmed/24475062
http://dx.doi.org/10.1371/journal.pone.0085943
work_keys_str_mv AT kovacsklaudia expressionandpropertiesofthehighlyalkalophilicphenylalanineammonialyaseofthermophilicrubrobacterxylanophilus
AT banoczigergely expressionandpropertiesofthehighlyalkalophilicphenylalanineammonialyaseofthermophilicrubrobacterxylanophilus
AT vargaandrea expressionandpropertiesofthehighlyalkalophilicphenylalanineammonialyaseofthermophilicrubrobacterxylanophilus
AT szaboizabella expressionandpropertiesofthehighlyalkalophilicphenylalanineammonialyaseofthermophilicrubrobacterxylanophilus
AT holczingerandras expressionandpropertiesofthehighlyalkalophilicphenylalanineammonialyaseofthermophilicrubrobacterxylanophilus
AT hornyanszkygabor expressionandpropertiesofthehighlyalkalophilicphenylalanineammonialyaseofthermophilicrubrobacterxylanophilus
AT zagyvaimre expressionandpropertiesofthehighlyalkalophilicphenylalanineammonialyaseofthermophilicrubrobacterxylanophilus
AT paizscsaba expressionandpropertiesofthehighlyalkalophilicphenylalanineammonialyaseofthermophilicrubrobacterxylanophilus
AT vertessybeatag expressionandpropertiesofthehighlyalkalophilicphenylalanineammonialyaseofthermophilicrubrobacterxylanophilus
AT poppelaszlo expressionandpropertiesofthehighlyalkalophilicphenylalanineammonialyaseofthermophilicrubrobacterxylanophilus