Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Aug;176(15):4511–4517. doi: 10.1128/jb.176.15.4511-4517.1994

Octopine and nopaline oxidases from Ti plasmids of Agrobacterium tumefaciens: molecular analysis, relationship, and functional characterization.

H Zanker 1, G Lurz 1, U Langridge 1, P Langridge 1, D Kreusch 1, J Schröder 1
PMCID: PMC196269  PMID: 8045881

Abstract

The occ and noc regions of pTiAch5 (octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of octopine and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The octopine oxidase had high activity with octopine (Km = 1 mM) and barely detectable activity with nopaline. The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity.

Full text

PDF
4511

Images in this article

4514Image
on p.4514

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bairoch A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 1992 May 11;20 (Suppl):2013–2018. doi: 10.1093/nar/20.suppl.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bevan M., Barnes W. M., Chilton M. D. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res. 1983 Jan 25;11(2):369–385. doi: 10.1093/nar/11.2.369. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. De Greve H., Decraemer H., Seurinck J., Van Montagu M., Schell J. The functional organization of the octopine Agrobacterium tumefaciens plasmid pTiB6s3. Plasmid. 1981 Sep;6(2):235–248. doi: 10.1016/0147-619x(81)90069-x. [DOI] [PubMed] [Google Scholar]
  5. De Greve H., Dhaese P., Seurinck J., Lemmers M., Van Montagu M., Schell J. Nucleotide sequence and transcript map of the Agrobacterium tumefaciens Ti plasmid-encoded octopine synthase gene. J Mol Appl Genet. 1982;1(6):499–511. [PubMed] [Google Scholar]
  6. De Vos G., De Beuckeleer M., Van Montagu M., Schell J. Restriction endonuclease mapping of the octopine tumor-inducing plasmid pTiAch5 of Agrobacterium tumefaciens. Plasmid. 1981 Sep;6(2):249–253. doi: 10.1016/0147-619x(81)90070-6. [DOI] [PubMed] [Google Scholar]
  7. Depicker A., Stachel S., Dhaese P., Zambryski P., Goodman H. M. Nopaline synthase: transcript mapping and DNA sequence. J Mol Appl Genet. 1982;1(6):561–573. [PubMed] [Google Scholar]
  8. Habeeb L. F., Wang L., Winans S. C. Transcription of the octopine catabolism operon of the Agrobacterium tumor-inducing plasmid pTiA6 is activated by a LysR-type regulatory protein. Mol Plant Microbe Interact. 1991 Jul-Aug;4(4):379–385. doi: 10.1094/mpmi-4-379. [DOI] [PubMed] [Google Scholar]
  9. Marincs F., White D. W. Nopaline causes a conformational change in the NocR regulatory protein-nocR promoter complex of Agrobacterium tumefaciens Ti plasmid pTiT37. Mol Gen Genet. 1993 Oct;241(1-2):65–72. doi: 10.1007/BF00280202. [DOI] [PubMed] [Google Scholar]
  10. Nakamura K., Inouye M. Construction of versatile expression cloning vehicles using the lipoprotein gene of Escherichia coli. EMBO J. 1982;1(6):771–775. doi: 10.1002/j.1460-2075.1982.tb01244.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nebert D. W., Gonzalez F. J. P450 genes: structure, evolution, and regulation. Annu Rev Biochem. 1987;56:945–993. doi: 10.1146/annurev.bi.56.070187.004501. [DOI] [PubMed] [Google Scholar]
  12. Otten L., Canaday J., Gérard J. C., Fournier P., Crouzet P., Paulus F. Evolution of agrobacteria and their Ti plasmids--a review. Mol Plant Microbe Interact. 1992 Jul-Aug;5(4):279–287. doi: 10.1094/mpmi-5-279. [DOI] [PubMed] [Google Scholar]
  13. Pearson W. R., Lipman D. J. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988 Apr;85(8):2444–2448. doi: 10.1073/pnas.85.8.2444. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Sans N., Schindler U., Schröder J. Ornithine cyclodeaminase from Ti plasmid C58: DNA sequence, enzyme properties and regulation of activity by arginine. Eur J Biochem. 1988 Apr 5;173(1):123–130. doi: 10.1111/j.1432-1033.1988.tb13975.x. [DOI] [PubMed] [Google Scholar]
  15. Sans N., Schröder G., Schröder J. The Noc region of Ti plasmid C58 codes for arginase and ornithine cyclodeaminase. Eur J Biochem. 1987 Aug 17;167(1):81–87. doi: 10.1111/j.1432-1033.1987.tb13306.x. [DOI] [PubMed] [Google Scholar]
  16. Schindler U., Sans N., Schröder J. Ornithine cyclodeaminase from octopine Ti plasmid Ach5: identification, DNA sequence, enzyme properties, and comparison with gene and enzyme from nopaline Ti plasmid C58. J Bacteriol. 1989 Feb;171(2):847–854. doi: 10.1128/jb.171.2.847-854.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Schrell A., Alt-Moerbe J., Lanz T., Schroeder J. Arginase of Agrobacterium Ti plasmid C58. DNA sequence, properties, and comparison with eucaryotic enzymes. Eur J Biochem. 1989 Oct 1;184(3):635–641. doi: 10.1111/j.1432-1033.1989.tb15060.x. [DOI] [PubMed] [Google Scholar]
  18. Schrell A., Schröder J. Characterization of a nopaline-induced gene for a 40 kDa protein in the nopaline catabolic (noc) region of Ti plasmid pTiC58. Biochim Biophys Acta. 1993 Sep 23;1174(3):303–304. doi: 10.1016/0167-4781(93)90204-q. [DOI] [PubMed] [Google Scholar]
  19. Thompson J., Donkersloot J. A. N-(carboxyalkyl)amino acids: occurrence, synthesis, and functions. Annu Rev Biochem. 1992;61:517–557. doi: 10.1146/annurev.bi.61.070192.002505. [DOI] [PubMed] [Google Scholar]
  20. Valdivia R. H., Wang L., Winans S. C. Characterization of a putative periplasmic transport system for octopine accumulation encoded by Agrobacterium tumefaciens Ti plasmid pTiA6. J Bacteriol. 1991 Oct;173(20):6398–6405. doi: 10.1128/jb.173.20.6398-6405.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Zanker H., von Lintig J., Schröder J. Opine transport genes in the octopine (occ) and nopaline (noc) catabolic regions in Ti plasmids of Agrobacterium tumefaciens. J Bacteriol. 1992 Feb;174(3):841–849. doi: 10.1128/jb.174.3.841-849.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. von Lintig J., Zanker H., Schröder J. Positive regulators of opine-inducible promoters in the nopaline and octopine catabolism regions of Ti plasmids. Mol Plant Microbe Interact. 1991 Jul-Aug;4(4):370–378. doi: 10.1094/mpmi-4-370. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES