Cloning, sequencing and heterologous expression of the gene for lupanine hydroxylase, a quinocytochrome c from a Pseudomonas sp.
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 M (r) 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 Ca(2+) 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.
Institute of Biological Sciences, University of Wales, Aberystwyth, Ceredigion SY23 3DD, Wales, U.K. dvh@aber.ac.uk
David J
DJ
Hopper
Mustak A
MA
Kaderbhai
Shirley A
SA
Marriott
Michael
M
Young
Jerzy
J
Rogozinski
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics* / Q000378:metabolism*; D045542:PQQ Cofactor; D021382:Protein Sorting Signals; D021381:Protein Transport; D011549:Pseudomonas / Q000235:genetics*; D015363:Quinolones / Q000737:chemistry / Q000378:metabolism; D011809:Quinones / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D021382:Protein Sorting Signals; D015363:Quinolones; D011809:Quinones; D011994:Recombinant Proteins; D045542:PQQ Cofactor; C023929:lupanine hydroxylase; D000587:Oxidoreductases Acting on CH-NH Group Donors; D002118:Calcium