Thelin Operon in Pseudomonas Incognita: Cloning, Nucleotide Sequence and Deduced Amino Acid Sequence of Cytochrome P-450lin and Other Enzymes Involved in the Catabolism of Linalool
Ropp, Jerry Dezz
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https://hdl.handle.net/2142/72355
Description
Title
Thelin Operon in Pseudomonas Incognita: Cloning, Nucleotide Sequence and Deduced Amino Acid Sequence of Cytochrome P-450lin and Other Enzymes Involved in the Catabolism of Linalool
Author(s)
Ropp, Jerry Dezz
Issue Date
1993
Doctoral Committee Chair(s)
Sligar, Stephen G.
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Microbiology
Chemistry, Biochemistry
Abstract
Cytochrome P-450 catalyzed reactions are extremely important in the metabolic pathways of both pro- and eucaryotes. Detailed mechanistic understanding of this superfamily, however, has been hampered by the availability of only one well characterized procaryotic system. Preliminary characterization of cytochrome P-450lin (P-450lin), the enzyme responsible for the 8-methyl hydroxylation of linalool as the first committed step of Pseudomonas incognita's utilization of that substrate as its sole carbon source, indicates its importance in expanding the foundation of the P-450 superfamily. Paramount to detailed mechanistic dissection, however, is the availability of a genetic handle, reagent quantities of pure enzyme and a reliable tertiary structure. Herein, we describe the realization of the first two goals which should aid in the completion of the third. Utilizing a Polymerase Chain Reaction-based cloning strategy based on the P-450lin NH$\sb2$-terminal and tryptic-fragment amino acid sequence, the cytochrome P-450lin operon (lin) was cloned. Four overlapping clones representing 10.8 kb of novel sequence were isolated and sequenced. Eight open reading frames were found in this sequence. The GenBank, EMBL, and SwissProt databases were searched in order to identify the translated sequences. Their identities based on the database search are as follows: (1) inconclusive, (2) hydratase, (3) P-450lin, (4) regulatory, (5) linalool-8-aldehyde dehydrogenase, (6) linalool-8-alcohol dehydrogenase, (7) lin-redoxin and (8) lin-redoxin reductase. The identities of the P-450lin, lin-redoxin, lin-redoxin reductase and linalool-8-alcohol dehydrogenase were also confirmed by the presence of the anticipated NH$\sb2$-terminal amino acid sequence determined for these proteins previously. A putative promoter sequence has been tentatively identified preceding OPF3. An optimized amino acid sequence alignment of P-450lin with cytochrome P-450cam shows the two enzymes to have only 25% identity. Based on the low sequence identity between P-450lin and all other P-450 sequences ($<$30%), P-450lin has been identified as the sole member of a new prokaryotic P-450 family, CYP111, in accordance with the nomenclature guidelines for the P-450 superfamily.
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