Validation of Putative Enzymes from Genomic Projects:
Confirmation of Dehalogenation ARv2ctivity for Mycobacterial
Protein 579

D 2003

Validation of Putative Enzymes from Genomic Projects: Confirmation of Dehalogenation ARv2ctivity for Mycobacterial Protein 579

MONINCOVÁ, Marta; Yuji NAGATA; Zbyněk PROKOP; Soňa MARVANOVÁ; Jana SÝKOROVÁ et al.

Základní údaje

Originální název

Validation of Putative Enzymes from Genomic Projects: Confirmation of Dehalogenation ARv2ctivity for Mycobacterial Protein 579

Název česky

Validace Enzymů:Zjištění dehalogenační aktivity Mzkobakteriálního proteinu 579.

Autoři

MONINCOVÁ, Marta; Yuji NAGATA; Zbyněk PROKOP; Soňa MARVANOVÁ; Jana SÝKOROVÁ; Masataka TSUDA a Jiří DAMBORSKÝ

Vydání

Manchester, UK, Management and Control of (Undesirable) Microorganisms, IBBS/IBRG Meeting Abstracts, s. 28-28, 2003

Nakladatel

IBBS/IBRG

Další údaje

Jazyk

angličtina

Typ výsledku

Stať ve sborníku

Obor

10600 1.6 Biological sciences

Stát vydavatele

Velká Británie a Severní Irsko

Utajení

není předmětem státního či obchodního tajemství

Označené pro přenos do RIV

Ano

Organizační jednotka

Přírodovědecká fakulta

Klíčová slova anglicky

dehalogenase; halogenated hydrocarbons; site-directed mutagenesis

Štítky

dehalogenase, halogenated hydrocarbons, site-directed mutagenesis

Změněno: 20. 6. 2008 13:14, prof. Mgr. Jiří Damborský, Dr.

Anotace

ORIG CZ

V originále

Haloalkane dehalogenases are the microbial enzymes which catalyse cleavage of the carbon-halogen bond. There are many microorganisms capable to dehalogenate halogenated hydrocarbons. Hydrolytic dehalogenating activity was detected also with a number of different mycobacteria. The putative haloalkane dehalogenase gene rv2579 was found in the complete genomic sequence of the human pathogen Mycobacterium tuberculosis H37Rv. Initially, computer modelling and site-directed mutagenesis experiments were conducted to confirm dehalogenating activity for Rv2579. The homology model of protein Rv2579 was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26. This analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six residues, mutations were introduced cumulatively into six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase family. Consequently the rv2579 gene was cloned from Mycobacterium bovis into Escherichia coli. Rv2579 protein was overproduced and purified to homogeneity. Rv2579 shows hydrolytic dehalogenating activity, further confirming the conclusions of the site-directed mutagenesis study. Detailed comparison of the catalytic properties of sixfold LinB mutant and Rv2579 is in progress. This comparison will validate applicability of reconstruction of an active site of an enzyme with putative function in an enzyme with known function.

Česky

xxxx

Citovat

MONINCOVÁ, Marta; Yuji NAGATA; Zbyněk PROKOP; Soňa MARVANOVÁ; Jana SÝKOROVÁ; Masataka TSUDA a Jiří DAMBORSKÝ. Validation of Putative Enzymes from Genomic Projects: Confirmation of Dehalogenation ARv2ctivity for Mycobacterial Protein 579. In Management and Control of (Undesirable) Microorganisms, IBBS/IBRG Meeting Abstracts. Manchester, UK: IBBS/IBRG, 2003, s. 28.

@inproceedings{491513,
   author = {Monincová, Marta and Nagata, Yuji and Prokop, Zbyněk and Marvanová, Soňa and Sýkorová, Jana and Tsuda, Masataka and Damborský, Jiří},
   address = {Manchester, UK},
   booktitle = {Management and Control of (Undesirable) Microorganisms, IBBS/IBRG Meeting Abstracts},
   keywords = {dehalogenase; halogenated hydrocarbons; site-directed mutagenesis},
   language = {eng},
   location = {Manchester, UK},
   pages = {28-28},
   publisher = {IBBS/IBRG},
   title = {Validation of Putative Enzymes from Genomic Projects: Confirmation of Dehalogenation ARv2ctivity for Mycobacterial Protein 579},
   year = {2003}
}

TY  - CONF
ID  - 491513
AU  - Monincová, Marta - Nagata, Yuji - Prokop, Zbyněk - Marvanová, Soňa - Sýkorová, Jana - Tsuda, Masataka - Damborský, Jiří
PY  - 2003
TI  - Validation of Putative Enzymes from Genomic Projects: Confirmation of Dehalogenation ARv2ctivity for Mycobacterial Protein 579
PB  - IBBS/IBRG
CY  - Manchester, UK
KW  - dehalogenase
KW  - halogenated hydrocarbons
KW  - site-directed mutagenesis
N2  - Haloalkane dehalogenases are the microbial enzymes which catalyse cleavage of the carbon-halogen bond. There are many microorganisms capable to dehalogenate halogenated hydrocarbons. Hydrolytic dehalogenating activity was detected also with a number of different mycobacteria. The putative haloalkane dehalogenase gene rv2579 was found in the complete genomic sequence of the human pathogen Mycobacterium tuberculosis H37Rv. Initially, computer modelling and site-directed mutagenesis experiments were conducted to confirm dehalogenating activity for Rv2579. The homology model of protein Rv2579 was compared with the crystal structure of haloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26. This analysis revealed that 6 of 19 amino acid residues which form an active site and entrance tunnel are different in LinB and Rv2579. To characterize the effect of replacement of these six residues, mutations were introduced cumulatively into six amino acid residues of LinB. The sixfold mutant, which was supposed to have the active site of Rv2579, exhibited dehalogenase activity with the haloalkanes tested, confirming that Rv2579 is a member of the haloalkane dehalogenase family. Consequently the rv2579 gene was cloned from Mycobacterium bovis into Escherichia coli. Rv2579 protein was overproduced and purified to homogeneity. Rv2579 shows hydrolytic dehalogenating activity, further confirming the conclusions of the site-directed mutagenesis study. Detailed comparison of the catalytic properties of sixfold LinB mutant and Rv2579 is in progress. This comparison will validate applicability of reconstruction of an active site of an enzyme with putative function in an enzyme with known function.
ER  -

MONINCOVÁ, Marta; Yuji NAGATA; Zbyněk PROKOP; Soňa MARVANOVÁ; Jana SÝKOROVÁ; Masataka TSUDA a Jiří DAMBORSKÝ. Validation of Putative Enzymes from Genomic Projects: Confirmation of Dehalogenation ARv2ctivity for Mycobacterial Protein 579. In \textit{Management and Control of (Undesirable) Microorganisms, IBBS/IBRG Meeting Abstracts}. Manchester, UK: IBBS/IBRG, 2003, s.~28.

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