DOI: 10.1111/j.1432-1033.1987.tb13704.x. The role of the N‐terminus of the large subunit of ribulose‐bisphosphate carboxylase investigated by construction and expression of chimaeric genes

The role of the N‐terminus of the large subunit of ribulose‐bisphosphate carboxylase investigated by construction and expression of chimaeric genes

10.1111/j.1432-1033.1987.tb13704.x

Crossref journal-article

Wiley

European Journal of Biochemistry (311)

Abstract

The genes for the large and small subunits of ribulose bisphosphate carboxylase/oxygenase (Rubisco) from Anacystis nidulans have been expressed in Escherichia coli under the control of the lac promoter to produce active enzyme. The enzyme can be purified from the cells to yield up to 200 mg Rubisco/l cultured bacteria, and is indistinguishable from the enzyme extracted from A. nidulans. In order to investigate the role of the N‐terminus of the large subunit in catalysis, chimaeric genes were constructed where the DNA coding for the 12 N‐terminal amino acids in A. nidulans was replaced by DNA encoding the equivalent, but poorly conserved, region of either the wheat or maize large subunit. These genes, in constructs also containing the gene for the A. nidulans small subunit, were expressed in E. coli and produced enzymes with similar catalytic properties to the wild‐type Rubisco of A. nidulans. In contrast, when the N‐terminal region of the large subunit was replaced by unrelated amino acids encoded by the pUC8 polylinker, enzyme activity of the expressed protein was reduced by 90% under standard assay conditions, due to an approximately tenfold rise in the Km for ribulose 1,5‐bisphosphate. This confirms that the N‐terminus of the large subunit has a function in catalysis, either directly in substrate binding or in maintaining the integrity of the active site.

Bibliography

KETTLEBOROUGH, C. A., PARRY, M. A. J., BURTON, S., GUTTERIDGE, S., KEYS, A. J., & PHILLIPS, A. L. (1987). The role of the Nâterminus of the large subunit of ribuloseâbisphosphate carboxylase investigated by construction and expression of chimaeric genes. European Journal of Biochemistry, 170(1â2), 335â342. Portico.

Authors 6

Catherine A. KETTLEBOROUGH (first)
Martin A. J. PARRY (additional)
Steven BURTON (additional)
Steven GUTTERIDGE (additional)
Alfred J. KEYS (additional)
Andrew L. PHILLIPS (additional)

References 35 Referenced 23

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{'key': 'e_1_2_3_22_2', 'volume-title': 'Molecular cloning: a laboratory manual', 'author': 'Maniatis T.', 'year': '1982'} / Molecular cloning: a laboratory manual by Maniatis T. (1982)
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Dates

Type	When
Created	20 years, 5 months ago (March 3, 2005, 8:52 p.m.)
Deposited	2 years ago (Aug. 30, 2023, 3:54 p.m.)
Indexed	3 weeks, 5 days ago (Aug. 6, 2025, 9:46 a.m.)
Issued	37 years, 9 months ago (Dec. 1, 1987)
Published	37 years, 9 months ago (Dec. 1, 1987)
Published Online	20 years, 5 months ago (March 3, 2005)
Published Print	37 years, 9 months ago (Dec. 1, 1987)

Funders 0

None

BibTeX

@article{KETTLEBOROUGH_1987, title={The role of the N‐terminus of the large subunit of ribulose‐bisphosphate carboxylase investigated by construction and expression of chimaeric genes}, volume={170}, ISSN={1432-1033}, url={http://dx.doi.org/10.1111/j.1432-1033.1987.tb13704.x}, DOI={10.1111/j.1432-1033.1987.tb13704.x}, number={1–2}, journal={European Journal of Biochemistry}, publisher={Wiley}, author={KETTLEBOROUGH, Catherine A. and PARRY, Martin A. J. and BURTON, Steven and GUTTERIDGE, Steven and KEYS, Alfred J. and PHILLIPS, Andrew L.}, year={1987}, month=dec, pages={335–342} }

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