Molecular cloning, sequencing, expression, and site-directed mutagenesis of the 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase gene from Arthrobacter spec. Rü61a.
The ring cleaving enzyme 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD)) of Arthrobacter spec. Rü61a 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 1H-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 alpha/beta 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.
Betz
A
A
Institute of Microbiology, University of Hohenheim, Stuttgart, Germany.
Facey
S J
SJ
Hauer
B
B
Tshisuaka
B
B
Lingens
F
F
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001673:Biodegradation, Environmental; D003001:Cloning, Molecular; D049308:Dioxygenases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010105:Oxygenases / Q000096:biosynthesis / Q000235:genetics*; D011798:Quinaldines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D011798:Quinaldines; D011994:Recombinant Proteins; D010105:Oxygenases; C101932:1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (decyclizing, CO-forming); D049308:Dioxygenases