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Cloning, purification and characterisation of cystathionine gamma-synthase from Nicotiana tabacum.

Title data

Clausen, Tim ; Wahl, Markus C. ; Messerschmidt, Albrecht ; Huber, Robert ; Fuhrmann, Jens C. ; Laber, Bernd ; Streber, Wolfgang ; Steegborn, Clemens:
Cloning, purification and characterisation of cystathionine gamma-synthase from Nicotiana tabacum.
In: Biological Chemistry. Vol. 380 (October 1999) Issue 10 . - pp. 1237-1242.
ISSN 1431-6730
DOI: https://doi.org/10.1515/BC.1999.157

Abstract in another language

Cystathionine gamma-synthase, the enzyme catalysing the first reaction specific for methionine biosynthesis, has been cloned from Nicotiana tabacum, overexpressed in Escherichia coli and purified to homogeneity. The recombinant cystathionine gamma-synthase catalyses the pyridoxal 5'-phosphate dependent formation of L-cystathionine from L-homoserine phosphate and L-cysteine with apparent Km-values of 7.1+/-3.1 mM and of 0.23+/-0.07 mM, respectively. The enzyme was irreversibly inhibited by DL-propargylglycine (Ki = 18 microM, k(inact) = 0.56 min(-1)), while the homoserine phosphate analogues 3-(phosphonomethyl)pyridine-2-carboxylic acid, 4-(phosphonomethyl)pyridine-2-carboxylic acid, Z-3-(2-phosphonoethen-1-yl)pyridine-2-carboxylic acid, and DL-E-2-amino-5-phosphono-3-pentenoic acid acted as reversible competitive inhibitors with Ki values of 0.20, 0.30, 0.45, and 0.027 mM, respectively. In combination these results suggest a ping-pong mechanism for the cystathionine gamma-synthase reaction, with homoserine phosphate binding to the enzyme first. Large single crystals of cystathionine gamma-synthase diffracting to beyond 2.7 A resolution were obtained by the sitting drop vapour diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell constants a = 120.0 A, b = 129.5 A, c = 309.8 A, corresponding to two tetramers per asymmetric unit.

Further data

Item Type: Article in a journal
Refereed: Yes
Additional notes: PubMed-ID: 10595588
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry > Chair Biochemistry - Univ.-Prof. Dr. Clemens Steegborn
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry
Result of work at the UBT: No
DDC Subjects: 500 Science > 540 Chemistry
Date Deposited: 13 Apr 2015 06:41
Last Modified: 08 Feb 2016 08:36
URI: https://eref.uni-bayreuth.de/id/eprint/10004