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. 1997 May;114(1):131–136. doi: 10.1104/pp.114.1.131

Mutations in the small subunit of ribulose-1,5-bisphosphate carboxylase/ oxygenase increase the formation of the misfire product xylulose-1,5-bisphosphate.

R Flachmann 1, G Zhu 1, R G Jensen 1, H J Bohnert 1
PMCID: PMC158286  PMID: 9159945

Abstract

The small subunit (S) increases the catalytic efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) by stabilizing the active sites generated by four large subunit (L) dimers. This stabilization appears to be due to an influence of S on the reaction intermediate 2,3-enediol, which is formed after the abstraction of a proton from the substrate ribulose-1,5-bisphosphate. We tested the functional significance of residues that are conserved among most species in the carboxy-terminal part of S and analyzed their influence on the kinetic parameters of Synechococcus holoenzymes. The replacements in S (F92S, Q99G, and P108L) resulted in catalytic activities ranging from 95 to 43% of wild type. The specificity factors for the three mutant enzymes were little affected (90-96% of wild type), but Km(CO2) values increased 0.5- to 2-fold. Mutant enzymes with replacements Q99G and P108L showed increased mis-protonation, relative to carboxylation, of the 2,3-enediol intermediate, forming 2 to 3 times more xylulose-1,5-bisphosphate per ribulose-1,5-bisphosphate utilized than wild-type or F92S enzymes. The results suggest that specific alterations of the L/S interfaces and of the hydrophobic core of S are transmitted to the active site by long-range interactions. S interactions with L may restrict the flexibility of active-site residues in L.

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Selected References

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  1. Andrews T. J. Catalysis by cyanobacterial ribulose-bisphosphate carboxylase large subunits in the complete absence of small subunits. J Biol Chem. 1988 Sep 5;263(25):12213–12219. [PubMed] [Google Scholar]
  2. Eilenberg H., Beer S., Gepstein S., Geva N., Tadmor O., Zilberstein A. Variability in Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Small Subunits and Carboxylation Activity in Fern Gametophytes Grown under Different Light Spectra. Plant Physiol. 1991 Jan;95(1):298–304. doi: 10.1104/pp.95.1.298. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Flachmann R., Bohnert H. J. Replacement of a conserved arginine in the assembly domain of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit interferes with holoenzyme formation. J Biol Chem. 1992 May 25;267(15):10576–10582. [PubMed] [Google Scholar]
  4. Hartman F. C., Harpel M. R. Structure, function, regulation, and assembly of D-ribulose-1,5-bisphosphate carboxylase/oxygenase. Annu Rev Biochem. 1994;63:197–234. doi: 10.1146/annurev.bi.63.070194.001213. [DOI] [PubMed] [Google Scholar]
  5. Knight S., Andersson I., Brändén C. I. Crystallographic analysis of ribulose 1,5-bisphosphate carboxylase from spinach at 2.4 A resolution. Subunit interactions and active site. J Mol Biol. 1990 Sep 5;215(1):113–160. doi: 10.1016/S0022-2836(05)80100-7. [DOI] [PubMed] [Google Scholar]
  6. Larson E. M., Larimer F. W., Hartman F. C. Mechanistic insights provided by deletion of a flexible loop at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase. Biochemistry. 1995 Apr 11;34(14):4531–4537. doi: 10.1021/bi00014a005. [DOI] [PubMed] [Google Scholar]
  7. Lee B. G., Read B. A., Tabita F. R. Catalytic properties of recombinant octameric, hexadecameric, and heterologous cyanobacterial/bacterial ribulose- 1,5-bisphosphate carboxylase/oxygenase. Arch Biochem Biophys. 1991 Dec;291(2):263–269. doi: 10.1016/0003-9861(91)90133-4. [DOI] [PubMed] [Google Scholar]
  8. Lee B., Berka R. M., Tabita F. R. Mutations in the small subunit of cyanobacterial ribulose-bisphosphate carboxylase/oxygenase that modulate interactions with large subunits. J Biol Chem. 1991 Apr 25;266(12):7417–7422. [PubMed] [Google Scholar]
  9. Newman J., Branden C. I., Jones T. A. Structure determination and refinement of ribulose 1,5-bisphosphate carboxylase/oxygenase from Synechococcus PCC6301. Acta Crystallogr D Biol Crystallogr. 1993 Nov 1;49(Pt 6):548–560. doi: 10.1107/S090744499300530X. [DOI] [PubMed] [Google Scholar]
  10. Newman J., Gutteridge S. Structure of an effector-induced inactivated state of ribulose 1,5-bisphosphate carboxylase/oxygenase: the binary complex between enzyme and xylulose 1,5-bisphosphate. Structure. 1994 Jun 15;2(6):495–502. doi: 10.1016/s0969-2126(00)00050-2. [DOI] [PubMed] [Google Scholar]
  11. Newman J., Gutteridge S. The X-ray structure of Synechococcus ribulose-bisphosphate carboxylase/oxygenase-activated quaternary complex at 2.2-A resolution. J Biol Chem. 1993 Dec 5;268(34):25876–25886. [PubMed] [Google Scholar]
  12. Pierce J., Andrews T. J., Lorimer G. H. Reaction intermediate partitioning by ribulose-bisphosphate carboxylases with differing substrate specificities. J Biol Chem. 1986 Aug 5;261(22):10248–10256. [PubMed] [Google Scholar]
  13. Pierce J., Tolbert N. E., Barker R. Interaction of ribulosebisphosphate carboxylase/oxygenase with transition-state analogues. Biochemistry. 1980 Mar 4;19(5):934–942. doi: 10.1021/bi00546a018. [DOI] [PubMed] [Google Scholar]
  14. Read B. A., Tabita F. R. A hybrid ribulosebisphosphate carboxylase/oxygenase enzyme exhibiting a substantial increase in substrate specificity factor. Biochemistry. 1992 Jun 23;31(24):5553–5560. doi: 10.1021/bi00139a018. [DOI] [PubMed] [Google Scholar]
  15. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Serianni A. S., Pierce J., Barker R. Carbon-13-enriched carbohydrates: preparation of triose, tetrose, and pentose phosphates. Biochemistry. 1979 Apr 3;18(7):1192–1199. doi: 10.1021/bi00574a012. [DOI] [PubMed] [Google Scholar]
  17. Shinozaki K., Yamada C., Takahata N., Sugiura M. Molecular cloning and sequence analysis of the cyanobacterial gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci U S A. 1983 Jul;80(13):4050–4054. doi: 10.1073/pnas.80.13.4050. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Smrcka A. V., Ramage R. T., Bohnert H. J., Jensen R. G. Purification and characterization of large and small subunits of ribulose 1,5-bisphosphate carboxylase expressed separately in Escherichia coli. Arch Biochem Biophys. 1991 Apr;286(1):6–13. doi: 10.1016/0003-9861(91)90002-z. [DOI] [PubMed] [Google Scholar]
  19. Voordouw G., De Vries P. A., Van den Berg W. A., De Clerck E. P. Site-directed mutagenesis of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from Anacystis nidulans. Eur J Biochem. 1987 Mar 16;163(3):591–598. doi: 10.1111/j.1432-1033.1987.tb10908.x. [DOI] [PubMed] [Google Scholar]
  20. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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