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Molecular cloning, sequencing, expression, and site‐directed mutagenesis of the 1 H ‐3‐hydroxy‐4‐oxoquinaldine 2,4‐dioxygenase gene from Arthrobacter spec. R?61a
Author(s) -
Betz Andrea,
Facey Sandra J.,
Hauer Bernhard,
Tshisuaka Barbara,
Lingens Franz
Publication year - 2000
Publication title -
journal of basic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.58
H-Index - 54
eISSN - 1521-4028
pISSN - 0233-111X
DOI - 10.1002/(sici)1521-4028(200002)40:1<7::aid-jobm7>3.0.co;2-5
Subject(s) - dioxygenase , biochemistry , pseudomonas putida , chemistry , site directed mutagenesis , active site , mutagenesis , mutant , stereochemistry , enzyme , gene , biology
The ring cleaving enzyme 1 H ‐3‐hydroxy‐4‐oxoquinaldine 2,4‐dioxygenase (HOD) of Arthrobacter spec. R?61a is part of the quinaldine degradation pathway. Carbon monoxide and N‐acetyl‐anthranilate are the products formed by dioxygenolytic cleavage of two C—C bonds in the substrate's pyridine ring. The gene coding for HOD was cloned and sequenced. An isoelectric point of pH 5.40 and a molecular mass of 31,838 Da was deduced from the sequence. HOD is shown to be remarkably similar to 1 H ‐3‐hydroxy‐4‐oxoquinoline 2,4‐dioxygenase (QDO) of Pseudomonas putida 33/1, but not to other dioxygenases described so far. Consensus regions indicative for any chromophoric cofactor or any catalytically relevant metal were not detected. Sequence comparisons and secondary structure predictions revealed HOD as a new member of the α / β hydrolase fold family. Expression in E. coli yielded recombinant catalytically active His‐tagged HOD. S101A and D233A, two mutants of HOD, were obtained by site‐directed mutagenesis. Since their residual activity is 43.1% and 62.6%, respectively, they probably are of no catalytic relevance although they might play a role in the interaction between enzyme and substrate.