Facey SJ

References (2)

Title : Molecular cloning, sequencing, expression, and site-directed mutagenesis of the 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase gene from Arthrobacter spec. Ru61a - Betz_2000_J.Basic.Microbiol_40_7
Author(s) : Betz A , Facey SJ , Hauer B , Tshisuaka B , Lingens F
Ref : J Basic Microbiol , 40 :7 , 2000
Abstract : The ring cleaving enzyme 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD)) of Arthrobacter spec. R61a 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.
ESTHER : Betz_2000_J.Basic.Microbiol_40_7
PubMedSearch : Betz_2000_J.Basic.Microbiol_40_7
PubMedID: 10746195
Gene_locus related to this paper: artsp-hod

Title : Cloning, sequencing and disruption of a bromoperoxidase-catalase gene in Streptomyces venezuelae: evidence that it is not required for chlorination in chloramphenicol biosynthesis - Facey_1996_Microbiology_142_657
Author(s) : Facey SJ , Gross F , Vining LC , Yang K , van Pee KH
Ref : Microbiology , 142 :657 , 1996
Abstract : Genomic DNA libraries of Streptomyces venezuelae ISP5230 and of a mutant blocked at the chlorination step of chloramphenicol biosynthesis were probed by hybridization with a synthetic oligonucleotide corresponding to the N-terminal amino acid sequence of a bromoperoxidase-catalase purified from the wild-type strain. Hybridizing fragments obtained from the two strains were cloned and sequenced. Analysis of the nucleotide sequences demonstrated that the fragments contained the same 1449 bp open reading frame with no differences in nucleotide sequence. The deduced polypeptide encoded 483 amino acids with a calculated M(r) of 54,200; the N-terminal sequence was identical to that of the bromoperoxidase-catalase purified from wild-type S. venezuelae. Comparison of the amino acid sequence predicted for the cloned bromoperoxidase-catalase gene (bca) with database protein sequences showed a significant similarity to a group of prokaryotic and eukaryotic catalases, but none to other peroxidases or haloperoxidases. Replacement of the bca gene in the wild-type strain of S. venezuelae with a copy disrupted by insertion of a DNA fragment encoding apramycin resistance did not prevent chloramphenicol production. The results suggest that the role of the enzyme in S. venezuelae is related to its activity as a catalase rather than as a halogenating agent.
ESTHER : Facey_1996_Microbiology_142_657
PubMedSearch : Facey_1996_Microbiology_142_657
PubMedID: 8868441