Molecular cloning, sequencing, expression, and site-directed mutagenesis of the 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase gene from Arthrobacter spec. Ru61a

The ring cleaving enzyme 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) (1)) of Arthrobacter spec. Ru61a is part of the quinaldine degradation path way. Carbon monoxide and N-acetyl-anthranilate are the products formed by d ioxygenolytic cleavage of two C-C bonds in the substrate's pyridine ring. T he 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-dioxyg enase (QDO) of Pseudomonas putida 33/1, but not to other dioxygenases descr ibed so far. Consensus regions indicative for any chromophoric cofactor or any catalytically relevant metal were not detected. Sequence comparisons an d secondary structure predictions revealed HOD as a new member of the alpha /beta hydrolase fold family. Expression in E. coli yielded recombinant cata lytically 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 alt hough they might play a role in the inter action between enzyme and substra te.