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

Michael Young, Jerry Rogozinski, Shirley A. Marriott, Mustak A. Kaderbhai, David J. Hopper

Research output: Contribution to journalArticlepeer-review

16 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)483-489
Number of pages7
JournalBiochemical Journal
Volume367
DOIs
Publication statusPublished - 16 Jul 2002

Fingerprint

Dive into the research topics of 'Cloning, sequencing and heterologous expression of the gene for lupanine hydroxylase, a quinocytochrome c from a Pseudomonas sp.'. Together they form a unique fingerprint.

Cite this