Purification and sequencing of a-Pinene Monooxyzgenase from 'Pseudomonas fluorescens' NCIMB 11671
Author(s)
Angie Colocousi.
Type
Thesis
Abstract
This thesis describes the purification and sequencing of a- pine ne monooxygenase from Pseudomonas fluorescens NCIMB 11671 which appears to be a novel flavin-iron monooxygenase. This enzyme catalyses the NADH linked oxygenation of a-pinene to the corresponding epoxide, is induced by a-p inene and requires FAD and ferrous iron for activity.
a-Pinene monooxygenase has been purified 40-fold by ion exchange chromatography and chromatofocusing and was estimated to be at least 90% pure by electrophoresis. The native molecular weight of the enzyme which consists of two identical subunits is 131, 000. It contains 2 moles of FAD/mole of enzyme and Is remarkably stable especially when stored in phosphate buffers containing glycerol. Complementation assays between a genomic library of P. fluorescens and mutants that were lacking the monooxygenase activity revealed a cosmid clone which contained the monooxygenase gene. Subcloning of the insert DNA further complementation and expression procedures showed that the a-pinene monooxygenase gene is located within a 5.3-kb Sma 1-Sma I fragment. A large open reading frame of l .8 kb, encoding for the monooxygenase, was located within this fragm ent. No regions of homology to the E. coli consensus sequence were found upstream the coding reg ion. However, the promoter region has strong homology to the nif-li ke promoters that have been shown to be regulated by the alternative cr54 factor. Possible FAD and NAD binding sites, as well as a Rieske type FeS centre were located at the amino-terminus, the middle, and proximal to the carboxy terminus of the deduced amino acid sequence of the monooxygenase gene respectively. Finally, a DNA sequence with striking similarity to proteins that belong to the NtrC activator family was located downstream the monooxygenase gene, and also two highly conserved NtrC activator binding sites were located Just upstream of the promoter region.
a-Pinene monooxygenase has been purified 40-fold by ion exchange chromatography and chromatofocusing and was estimated to be at least 90% pure by electrophoresis. The native molecular weight of the enzyme which consists of two identical subunits is 131, 000. It contains 2 moles of FAD/mole of enzyme and Is remarkably stable especially when stored in phosphate buffers containing glycerol. Complementation assays between a genomic library of P. fluorescens and mutants that were lacking the monooxygenase activity revealed a cosmid clone which contained the monooxygenase gene. Subcloning of the insert DNA further complementation and expression procedures showed that the a-pinene monooxygenase gene is located within a 5.3-kb Sma 1-Sma I fragment. A large open reading frame of l .8 kb, encoding for the monooxygenase, was located within this fragm ent. No regions of homology to the E. coli consensus sequence were found upstream the coding reg ion. However, the promoter region has strong homology to the nif-li ke promoters that have been shown to be regulated by the alternative cr54 factor. Possible FAD and NAD binding sites, as well as a Rieske type FeS centre were located at the amino-terminus, the middle, and proximal to the carboxy terminus of the deduced amino acid sequence of the monooxygenase gene respectively. Finally, a DNA sequence with striking similarity to proteins that belong to the NtrC activator family was located downstream the monooxygenase gene, and also two highly conserved NtrC activator binding sites were located Just upstream of the promoter region.
Version
Open Access
Date Issued
1994
Copyright Statement
Attribution NoDerivatives 4.0 International Licence (CC BY-ND)