Files in this item

FilesDescriptionFormat

application/pdf

application/pdf8823271.pdf (6MB)Restricted to U of Illinois
(no description provided)PDF

Description

Title:Sequence, Expression, and Mutagenesis of the Pseudomonas Putida Cytochrome P450(cam) Gene in Escherichia Coli
Author(s):Unger, Benjamin Peter
Doctoral Committee Chair(s):Sligar, Stephen G.
Department / Program:Biochemistry
Discipline:Biochemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Biology, Molecular
Chemistry, Biochemistry
Abstract:Cytochrome P450$\sb{\rm cam}$ catalyzes the stereospecific methylene hydroxylation of camphor to form 5-exo-hydroxycamphor and is encoded by the camC gene on the CAM plasmid of Pseudomonas putida, ATCC 17453. The cytochrome P450$\sb{\rm cam}$ structural gene has been cloned by mutant complementation in P. putida (Koga et al. 1985. Biochem. Biophys. Res. Commun. 130, 412-417). This investigation reports the complete nucleotide sequence of the camC gene along with 155 base pairs of 5$\sp\prime$ and approximately 790 base pairs of 3$\sp\prime$ flanking sequence. The gene encoding putidaredoxin reductase, camA, was located 22 nucleotides downstream from the camC gene. The camA gene initiated with a novel GUG codon, the first such initiator documented in Pseudomonas.
Upon comparison of the amino acid sequence derived from the camC gene sequence to the one obtained from the purified P450$\sb{\rm cam}$ enzyme, (Haniu et al. (1982). J. Biol. Chem. 257, 12664-12671) five differences were found. The most significant was the addition of Trp and a Thr residue between Val-54 and Arg-55, thereby increasing the amino acid numbering scheme by two after Val-54, bringing the total number of amino acids to 414. Other differences were: Gin-274 $\to$ Glu-276, Ser-359 $\to$ His-361, and Asn-405 $\to$ Asp-407. N-terminal amino acid sequence analysis of the cloned P450$\sb{\rm cam}$ enzyme expressed in Escherichia coli under the lac promoter showed a faithful translation of the hemeprotein, with the N-terminal Met removed by processing as found in P. putida.
In vitro site-directed mutagenesis of the cytochrome P450$\sb{\rm cam}$ heme axial ligand codon, Cys-357, was performed to determine the ligand's role in P450-dependent catalysis. The Cys-357 codon was replaced by either a His or a Ser codon. Purification of Cys357-His P450$\sb{\rm cam}$ and study of some of the enzyme's properties revealed it to be incompetent in camphor hydroxylation either in the reconstituted system with NADH or with exogenous oxidants. The mutant's redox potential, autoxidation rate, and NADH oxidation rate paralleled those determined for the P. putida enzyme.
Issue Date:1988
Type:Text
Description:219 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.
URI:http://hdl.handle.net/2142/70571
Other Identifier(s):(UMI)AAI8823271
Date Available in IDEALS:2014-12-15
Date Deposited:1988


This item appears in the following Collection(s)

Item Statistics