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. 2002 Oct 15;367(Pt 2):483–489. doi: 10.1042/BJ20020729

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

David J Hopper 1, Mustak A Kaderbhai 1, Shirley A Marriott 1, Michael Young 1, Jerzy Rogozinski 1
PMCID: PMC1222901  PMID: 12119046

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 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.

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Selected References

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  1. Altschul S. F., Madden T. L., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997 Sep 1;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anthony C., Ghosh M. The structure and function of the PQQ-containing quinoprotein dehydrogenases. Prog Biophys Mol Biol. 1998;69(1):1–21. doi: 10.1016/s0079-6107(97)00020-5. [DOI] [PubMed] [Google Scholar]
  3. Berks B. C. A common export pathway for proteins binding complex redox cofactors? Mol Microbiol. 1996 Nov;22(3):393–404. doi: 10.1046/j.1365-2958.1996.00114.x. [DOI] [PubMed] [Google Scholar]
  4. Chambers S. P., Prior S. E., Barstow D. A., Minton N. P. The pMTL nic- cloning vectors. I. Improved pUC polylinker regions to facilitate the use of sonicated DNA for nucleotide sequencing. Gene. 1988 Aug 15;68(1):139–149. doi: 10.1016/0378-1119(88)90606-3. [DOI] [PubMed] [Google Scholar]
  5. Cozier G. E., Anthony C. Structure of the quinoprotein glucose dehydrogenase of Escherichia coli modelled on that of methanol dehydrogenase from Methylobacterium extorquens. Biochem J. 1995 Dec 15;312(Pt 3):679–685. doi: 10.1042/bj3120679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ghosh M., Anthony C., Harlos K., Goodwin M. G., Blake C. The refined structure of the quinoprotein methanol dehydrogenase from Methylobacterium extorquens at 1.94 A. Structure. 1995 Feb 15;3(2):177–187. doi: 10.1016/s0969-2126(01)00148-4. [DOI] [PubMed] [Google Scholar]
  7. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  8. He M., Wilde A., Kaderbhai M. A. A simple single-step procedure for small-scale preparation of Escherichia coli plasmids. Nucleic Acids Res. 1990 Mar 25;18(6):1660–1660. doi: 10.1093/nar/18.6.1660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Hopper D. J., Rogozinski J. Redox potential of the haem c group in the quinocytochrome, lupanine hydroxylase, an enzyme located in the periplasm of a Pseudomonas sp. Biochim Biophys Acta. 1998 Mar 3;1383(1):160–164. doi: 10.1016/s0167-4838(97)00204-5. [DOI] [PubMed] [Google Scholar]
  10. Hopper D. J., Rogozinski J., Toczko M. Lupanine hydroxylase, a quinocytochrome c from an alkaloid-degrading Pseudomonas sp. Biochem J. 1991 Oct 1;279(Pt 1):105–109. doi: 10.1042/bj2790105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kaderbhai M. A., Ugochukwu C. C., Kelly S. L., Lamb D. C. Export of cytochrome P450 105D1 to the periplasmic space of Escherichia coli. Appl Environ Microbiol. 2001 May;67(5):2136–2138. doi: 10.1128/AEM.67.5.2136-2138.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Keitel T., Diehl A., Knaute T., Stezowski J. J., Höhne W., Görisch H. X-ray structure of the quinoprotein ethanol dehydrogenase from Pseudomonas aeruginosa: basis of substrate specificity. J Mol Biol. 2000 Apr 7;297(4):961–974. doi: 10.1006/jmbi.2000.3603. [DOI] [PubMed] [Google Scholar]
  13. Morgenstern B. DIALIGN 2: improvement of the segment-to-segment approach to multiple sequence alignment. Bioinformatics. 1999 Mar;15(3):211–218. doi: 10.1093/bioinformatics/15.3.211. [DOI] [PubMed] [Google Scholar]
  14. Nielsen H., Engelbrecht J., Brunak S., von Heijne G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 1997 Jan;10(1):1–6. doi: 10.1093/protein/10.1.1. [DOI] [PubMed] [Google Scholar]
  15. Oubrie A., Dijkstra B. W. Structural requirements of pyrroloquinoline quinone dependent enzymatic reactions. Protein Sci. 2000 Jul;9(7):1265–1273. doi: 10.1110/ps.9.7.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Oubrie A., Rozeboom H. J., Kalk K. H., Duine J. A., Dijkstra B. W. The 1.7 A crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat. J Mol Biol. 1999 Jun 4;289(2):319–333. doi: 10.1006/jmbi.1999.2766. [DOI] [PubMed] [Google Scholar]
  17. Oubrie Arthur, Rozeboom Henriëtte J., Kalk Kor H., Huizinga Eric G., Dijkstra Bauke W. Crystal structure of quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni: structural basis for substrate oxidation and electron transfer. J Biol Chem. 2001 Nov 19;277(5):3727–3732. doi: 10.1074/jbc.M109403200. [DOI] [PubMed] [Google Scholar]
  18. Rogoziński J. Molecular properties of the inducible lupanine hydroxylase from growing cultures of Pseudomonas lupanini. Acta Biochim Pol. 1975;22(1):57–66. [PubMed] [Google Scholar]
  19. Stoorvogel J., Kraayveld D. E., Van Sluis C. A., Jongejan J. A., De Vries S., Duine J. A. Characterization of the gene encoding quinohaemoprotein ethanol dehydrogenase of Comamonas testosteroni. Eur J Biochem. 1996 Feb 1;235(3):690–698. doi: 10.1111/j.1432-1033.1996.00690.x. [DOI] [PubMed] [Google Scholar]
  20. Thöny-Meyer L. Biogenesis of respiratory cytochromes in bacteria. Microbiol Mol Biol Rev. 1997 Sep;61(3):337–376. doi: 10.1128/mmbr.61.3.337-376.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Xia Z., Dai W., Zhang Y., White S. A., Boyd G. D., Mathews F. S. Determination of the gene sequence and the three-dimensional structure at 2.4 angstroms resolution of methanol dehydrogenase from Methylophilus W3A1. J Mol Biol. 1996 Jun 14;259(3):480–501. doi: 10.1006/jmbi.1996.0334. [DOI] [PubMed] [Google Scholar]
  22. de Jong G. A., Caldeira J., Sun J., Jongejan J. A., de Vries S., Loehr T. M., Moura I., Moura J. J., Duine J. A. Characterization of the interaction between PQQ and heme c in the quinohemoprotein ethanol dehydrogenase from Comamonas testosteroni. Biochemistry. 1995 Jul 25;34(29):9451–9458. doi: 10.1021/bi00029a021. [DOI] [PubMed] [Google Scholar]

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