Cloning, sequencing and heterologous expression of the gene for lupanine hydroxylase, a quinocytochrome c from a Pseudomonas sp.

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dc.contributor.author Young, Michael
dc.contributor.author Rogozinski, Jerry
dc.contributor.author Marriott, Shirley A.
dc.contributor.author Kaderbhai, Mustak A.
dc.contributor.author Hopper, David J.
dc.date.accessioned 2008-12-04T14:52:51Z
dc.date.available 2008-12-04T14:52:51Z
dc.date.issued 2002-07-16
dc.identifier.citation Young , M , Rogozinski , J , Marriott , S A , Kaderbhai , M A & Hopper , D J 2002 , ' Cloning, sequencing and heterologous expression of the gene for lupanine hydroxylase, a quinocytochrome c from a Pseudomonas sp. ' Biochemical Journal , vol 367 , pp. 483-489 . en
dc.identifier.issn 1470-8728
dc.identifier.other PURE: 94074
dc.identifier.other dspace: 2160/1352
dc.identifier.uri http://hdl.handle.net/2160/1352
dc.identifier.uri http://www.biochemj.org/bj/367/0483/3670483.pdf en
dc.description Hopper D., M.A. Kaderbhai, S.A. Marriott., M. Young, J. Rogoziński (2002). Cloning, sequencing and heterologous expression of the gene for lupanine hydroxylase, a quinocytochrome c from a Pseudomonas sp. Biochemical Journal 367, 483-489. Sponsorship: BBSRC RAE2008 en
dc.description.abstract The gene encoding the enzyme lupanine hydroxylase was isolated by PCR using chromosomal DNA from a lupanine-utilizing Pseudomonas sp. as template and primers based on the sequences of the N- and C-termini of the purified protein. The derived sequence for the mature gene product gave a protein with an Mr of 72256, in good agreement with the value found by SDS/PAGE of the pure enzyme, and contained the sequences of several peptides obtained after endoproteinase Lys-C digestion of the pure enzyme. The gene, under the transcriptional control of a phoA promotor and with the Escherichia coli alkaline phosphatase signal sequence, was expressed in E. coli containing a plasmid expressing the genes for cytochrome c maturation proteins constitutively. Haem-containing inactive protein in inclusion bodies was renatured and reactivated with pyrroloquinoline quinone (PQQ) and Ca2+ to give active enzyme. The lupanine hydroxylase (luh) gene coded for a protein with a cleavable 26-residue signal sequence at its N-terminus, required for the transport of the enzyme to its periplasmic location. Analysis of the protein sequence showed that it contains two domains, a large PQQ-binding N-terminal domain and a smaller cytochrome c C-terminal domain. Comparison of the derived sequence with those of other proteins showed considerable similarity with other quino(haemo)proteins, including alcohol dehydrogenases from a variety of bacteria. The PQQ-binding domain sequence contains W motifs, characteristic of the eight-bladed 'propeller' structure of methanol dehydrogenase, but lacks the unusual disulphide ring structure formed from two adjacent cysteines seen in this enzyme. The C-terminus shares some similarity with bacterial cytochrome c and includes the haem-binding consensus sequence CXXCH. en
dc.format.extent 7 en
dc.language.iso eng
dc.relation.ispartof Biochemical Journal en
dc.title Cloning, sequencing and heterologous expression of the gene for lupanine hydroxylase, a quinocytochrome c from a Pseudomonas sp. en
dc.type Text en
dc.type.publicationtype Article (Journal) en
dc.identifier.doi http://dx.doi.org/10.1042/BJ20020729
dc.contributor.institution Institute of Biological, Environmental and Rural Sciences en
dc.description.status Peer reviewed en


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