Protein engineering of haloalkane dehalogenase LinB:
reconstruction of active site and modification of entrance
tunnel

D 2006

Protein engineering of haloalkane dehalogenase LinB: reconstruction of active site and modification of entrance tunnel

MONINCOVÁ, Marta, Yuji NAGATA, Radka CHALOUPKOVÁ, Zbyněk PROKOP, Andrea FOŘTOVÁ et. al.

Basic information

Original name

Protein engineering of haloalkane dehalogenase LinB: reconstruction of active site and modification of entrance tunnel

Name in Czech

Proteinové inženýrství haloalkán dehalogenázy LinB: rekonstrukce aktivního místa a modifikace vstupního tunelu.

Authors

MONINCOVÁ, Marta (203 Czech Republic, belonging to the institution), Yuji NAGATA (392 Japan), Radka CHALOUPKOVÁ (203 Czech Republic, belonging to the institution), Zbyněk PROKOP (203 Czech Republic, belonging to the institution), Andrea FOŘTOVÁ (203 Czech Republic, belonging to the institution), Martina PAVLOVÁ (203 Czech Republic, belonging to the institution), Masataka TSUDA (392 Japan) and Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution)

Edition

1. vyd. Praha, Book of Abstracts, 10th International Symposium of the Genetics of Industrial Microorganisms, p. 160-161, 2 pp. 2006

Publisher

Institute of Microbiology ASCR

Other information

Language

English

Type of outcome

Stať ve sborníku

Field of Study

10600 1.6 Biological sciences

Country of publisher

Czech Republic

Confidentiality degree

není předmětem státního či obchodního tajemství

RIV identification code

RIV/00216224:14310/06:00017059

Organization unit

Faculty of Science

ISBN

80-239-7323-1

Keywords in English

haloalkane dehalogenase; protein engineering; entrance tunnel; active site

Abstract

ORIG CZ

V originále

Haloalkane dehalogenase LinB is an enzyme isolated from lindan degrading bacterium Sphingobium japonicum UT26. LinBs 3D structure [1], catalytic properties and substrate specificity are known and well studied. Thanks to these facts LinB is great target for protein engineering experiments. Firts experiment, reconstruction of active site, was based on 68% sequence identity with ORF rv2579 from Mycobacterium tuberculosis H37Rv genome. The homology model of protein Rv2579 was compared with the 3D structure of LinB. This analysis revealed that 6 out of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. The 6 different amino acids were cumulatively mutated in LinB. Final six-fold mutant was presumed to have active site and entrance tunnel of Rv2579 and exhibited dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase family. Consequently the M. tuberculosis gene rv2579 was cloned into Escherichia coli. Heterogously produced Rv2579 shows hydrolytic dehalogenating activity, further confirming the conclusions based on the site-directed mutagenesis study. This comparison validated applicability of reconstruction of an active site of an enzyme with putative function in an enzyme with known function. Second experiment, modification of entrance tunnel, was based on following observations. Comparison of three known 3D structures of haloalkane dehalogenases suggested that substrate specificity of these protein family could be significantly influenced by the size and shape of its entrance tunnel. Phylogenetic analysis revealed that residue lokalized in the mouth of the entrance tunnel is the most variable pocket rezidue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant LinB proteins carrying all possible mutations in position 177 were purified to homogenity and specific activities with set of 12 halogenated substrates were determined. Multivariate statistics [2] of activity data revealed that catalytic activity of mutant enzymes generaly increased with the indroduction of small and nonpolar aminoacids. Rational engineering is power tool to develop mutant enzymes with modified enzymatic properties rather than combinatorial screening. References: 1. Marek, J., Vevodova, J., Kuta-Smatanova, I., Nagata, Y., Svensson, L.A., Newman, J., Takagi, M., Damborsky, J.: Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26. Biochemistry, 2000. 39, 14082-14086. 2. Wold, S., Esbensen, K., Geladi, P.: Principal Component Analysis. Chemometrics and Intelligent Laboratory Systems, 1987. 2, 37-52.

In Czech

Haloalkán dehalogenáza LinB je enzym izolovaný z bakterie Sphingobium japonicum UT26, která je schopna degradovat lindan. Struktura LinB, katalytické vlastnosti a substrátová specifita jsou dobře znamy. proto je LinB vhodným kandidátem pro proteinové experimenty.

Links

LC06010, research and development project

Name: Centrum biokatalýzy a biotransformací

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

Citovat

MONINCOVÁ, Marta, Yuji NAGATA, Radka CHALOUPKOVÁ, Zbyněk PROKOP, Andrea FOŘTOVÁ, Martina PAVLOVÁ, Masataka TSUDA and Jiří DAMBORSKÝ. Protein engineering of haloalkane dehalogenase LinB: reconstruction of active site and modification of entrance tunnel. In Book of Abstracts, 10th International Symposium of the Genetics of Industrial Microorganisms. 1st ed. Praha: Institute of Microbiology ASCR, 2006, p. 160-161. ISBN 80-239-7323-1.

@inproceedings{692362,
   author = {Monincová, Marta and Nagata, Yuji and Chaloupková, Radka and Prokop, Zbyněk and Fořtová, Andrea and Pavlová, Martina and Tsuda, Masataka and Damborský, Jiří},
   address = {Praha},
   booktitle = {Book of Abstracts, 10th International Symposium of the Genetics of Industrial Microorganisms},
   edition = {1.},
   keywords = {haloalkane dehalogenase; protein engineering; entrance tunnel; active site},
   language = {eng},
   location = {Praha},
   isbn = {80-239-7323-1},
   pages = {160-161},
   publisher = {Institute of Microbiology ASCR},
   title = {Protein engineering of haloalkane dehalogenase LinB: reconstruction of active site and modification of entrance tunnel},
   year = {2006}
}

TY  - JOUR
ID  - 692362
AU  - Monincová, Marta - Nagata, Yuji - Chaloupková, Radka - Prokop, Zbyněk - Fořtová, Andrea - Pavlová, Martina - Tsuda, Masataka - Damborský, Jiří
PY  - 2006
TI  - Protein engineering of haloalkane dehalogenase LinB: reconstruction of active site and modification of entrance tunnel
PB  - Institute of Microbiology ASCR
CY  - Praha
SN  - 8023973231
KW  - haloalkane dehalogenase
KW  - protein engineering
KW  - entrance tunnel
KW  - active site
N2  - Haloalkane dehalogenase LinB is an enzyme isolated from lindan degrading bacterium Sphingobium japonicum UT26. LinBs 3D structure [1], catalytic properties and substrate specificity are known and well studied. Thanks to these facts LinB is great target for protein engineering experiments. Firts experiment, reconstruction of active site, was based on 68% sequence identity with ORF rv2579 from Mycobacterium tuberculosis H37Rv genome. The homology model of protein Rv2579 was compared with the 3D structure of LinB. This analysis revealed that 6 out of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. The 6 different amino acids were cumulatively mutated in LinB. Final six-fold mutant was presumed to have active site and entrance tunnel of Rv2579 and exhibited dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase family. Consequently the M. tuberculosis gene rv2579 was cloned into Escherichia coli. Heterogously produced Rv2579 shows hydrolytic dehalogenating activity, further confirming the conclusions based on the site-directed mutagenesis study. This comparison validated applicability of reconstruction of an active site of an enzyme with putative function in an enzyme with known function. Second experiment, modification of entrance tunnel, was based on following observations. Comparison of three known 3D structures of haloalkane dehalogenases suggested that substrate specificity of these protein family could be significantly influenced by the size and shape of its entrance tunnel. Phylogenetic analysis revealed that residue lokalized in the mouth of the entrance tunnel is the most variable pocket rezidue in haloalkane dehalogenase-like proteins with nine substitutions in 14 proteins. Mutant LinB proteins carrying all possible mutations in position 177 were purified to homogenity and specific activities with set of 12 halogenated substrates were determined. Multivariate statistics [2] of activity data revealed that catalytic activity of mutant enzymes generaly increased with the indroduction of small and nonpolar aminoacids. Rational engineering is power tool to develop mutant enzymes with modified enzymatic properties rather than combinatorial screening. References: 1. Marek, J., Vevodova, J., Kuta-Smatanova, I., Nagata, Y., Svensson, L.A., Newman, J., Takagi, M., Damborsky, J.: Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26. Biochemistry, 2000. 39, 14082-14086. 2. Wold, S., Esbensen, K., Geladi, P.: Principal Component Analysis. Chemometrics and Intelligent Laboratory Systems, 1987. 2, 37-52.
ER  -

MONINCOVÁ, Marta, Yuji NAGATA, Radka CHALOUPKOVÁ, Zbyněk PROKOP, Andrea FOŘTOVÁ, Martina PAVLOVÁ, Masataka TSUDA and Jiří DAMBORSKÝ. Protein engineering of haloalkane dehalogenase LinB: reconstruction of active site and modification of entrance tunnel. In \textit{Book of Abstracts, 10th International Symposium of the Genetics of Industrial Microorganisms}. 1st ed. Praha: Institute of Microbiology ASCR, 2006, p.~160-161. ISBN~80-239-7323-1.

Detailed Information on Publication Record