Modification of Activity and Specificity of Haloalkane
Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering
of its Entrance Tunnel

J 2003

Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of its Entrance Tunnel

CHALOUPKOVÁ, Radka, Jana SÝKOROVÁ, Zbyněk PROKOP, Andrea JESENSKÁ, Marta MONINCOVÁ et. al.

Basic information

Original name

Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of its Entrance Tunnel

Authors

CHALOUPKOVÁ, Radka (203 Czech Republic), Jana SÝKOROVÁ (203 Czech Republic), Zbyněk PROKOP (203 Czech Republic), Andrea JESENSKÁ (203 Czech Republic), Marta MONINCOVÁ (203 Czech Republic), Martina PAVLOVÁ (203 Czech Republic), Masataka TSUDA (392 Japan), Yuji NAGATA (392 Japan) and Jiří DAMBORSKÝ (203 Czech Republic, guarantor)

Edition

Journal of Biological Chemistry, 2003, 1083-351X

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10600 1.6 Biological sciences

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

URL

Impact factor

Impact factor: 6.482

RIV identification code

RIV/00216224:14310/03:00009370

Organization unit

Faculty of Science

UT WoS

000187480700080

Keywords in English

DEHALOGENASE MUTAGENESIS SPECIFICITY

Abstract

V originále

Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis: the residue partially blocks the entrance tunnel and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in fourteen proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, fifteen active protein variants were obtained suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterised by determination of specific activities with twelve halogenated substrates and steady-state kinetic parameters with two substrates. Effect of mutation on the enzyme activities varied dramatically with the structure of the substrates suggesting that extrapolation of one substrate to another may be misleading and a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and non-polar amino acid in the position 177. This result is consistent with the phylogenetic analysis which showed that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants. Although the approach did not result in mutant enzymes with entirely new capabilities it did result in improving the catalytic properties and expanding their substrate specificities.

Links

LN00A016, research and development project

Name: BIOMOLEKULÁRNÍ CENTRUM

Investor: Ministry of Education, Youth and Sports of the CR, Biomolecular Center

Citovat

CHALOUPKOVÁ, Radka, Jana SÝKOROVÁ, Zbyněk PROKOP, Andrea JESENSKÁ, Marta MONINCOVÁ, Martina PAVLOVÁ, Masataka TSUDA, Yuji NAGATA and Jiří DAMBORSKÝ. Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of its Entrance Tunnel. Journal of Biological Chemistry. 2003, vol. 278, No 52, p. 52622-52628. ISSN 1083-351X.

@article{491667,
   author = {Chaloupková, Radka and Sýkorová, Jana and Prokop, Zbyněk and Jesenská, Andrea and Monincová, Marta and Pavlová, Martina and Tsuda, Masataka and Nagata, Yuji and Damborský, Jiří},
   article_number = {52},
   keywords = {DEHALOGENASE MUTAGENESIS SPECIFICITY},
   language = {eng},
   issn = {1083-351X},
   journal = {Journal of Biological Chemistry},
   title = {Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of its Entrance Tunnel},
   url = {http://ncbr.chemi.muni.cz/~jiri/abstracts/jbc03c.html},
   volume = {278},
   year = {2003}
}

TY  - JOUR
ID  - 491667
AU  - Chaloupková, Radka - Sýkorová, Jana - Prokop, Zbyněk - Jesenská, Andrea - Monincová, Marta - Pavlová, Martina - Tsuda, Masataka - Nagata, Yuji - Damborský, Jiří
PY  - 2003
TI  - Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of its Entrance Tunnel
JF  - Journal of Biological Chemistry
VL  - 278
IS  - 52
SP  - 52622-52628
EP  - 52622-52628
SN  - 1083351X
KW  - DEHALOGENASE MUTAGENESIS SPECIFICITY
UR  - http://ncbr.chemi.muni.cz/~jiri/abstracts/jbc03c.html
N2  - Structural comparison of three different haloalkane dehalogenases suggested that substrate specificity of these bacterial enzymes could be significantly influenced by the size and shape of their entrance tunnels. The surface residue leucine 177 positioned at the tunnel opening of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 was selected for modification based on structural and phylogenetic analysis: the residue partially blocks the entrance tunnel and it is the most variable pocket residue in haloalkane dehalogenase-like proteins with nine substitutions in fourteen proteins. Mutant genes coding for proteins carrying all possible substitutions in position 177 were constructed by site-directed mutagenesis and heterologously expressed in Escherichia coli. In total, fifteen active protein variants were obtained suggesting a relatively high tolerance of the site for the introduction of mutations. Purified protein variants were kinetically characterised by determination of specific activities with twelve halogenated substrates and steady-state kinetic parameters with two substrates. Effect of mutation on the enzyme activities varied dramatically with the structure of the substrates suggesting that extrapolation of one substrate to another may be misleading and a systematic characterization of the protein variants with a number of substrates is essential. Multivariate analysis of activity data revealed that catalytic activity of mutant enzymes generally increased with the introduction of small and non-polar amino acid in the position 177. This result is consistent with the phylogenetic analysis which showed that glycine and alanine are the most commonly occurring amino acids in this position among haloalkane dehalogenases. The study demonstrates the advantages of using rational engineering to develop enzymes with modified catalytic properties and substrate specificities. The strategy of using site-directed mutagenesis to modify a specific entrance tunnel residue identified by structural and phylogenetic analyses, rather than combinatorial screening, generated a high percentage of viable mutants. Although the approach did not result in mutant enzymes with entirely new capabilities it did result in improving the catalytic properties and expanding their substrate specificities.
ER  -

CHALOUPKOVÁ, Radka, Jana SÝKOROVÁ, Zbyněk PROKOP, Andrea JESENSKÁ, Marta MONINCOVÁ, Martina PAVLOVÁ, Masataka TSUDA, Yuji NAGATA and Jiří DAMBORSKÝ. Modification of Activity and Specificity of Haloalkane Dehalogenase from Sphingomonas paucimobilis UT26 by Engineering of its Entrance Tunnel. \textit{Journal of Biological Chemistry}. 2003, vol.~278, No~52, p.~52622-52628. ISSN~1083-351X.

Detailed Information on Publication Record