Characterization of recombinant cysteine
synthase in Caenorhabditis elegans
R. Vozdek1, A. Hnizda1,
J. Krijt1, M. Kodicek2, V. Kozich1
1 Institute of Inherited Metabolic Diseases, First Faculty of Medicine, Charles University in Prague
2 Department of Biochemistry and Mikrobiology, Faculty of Food and Biochemical Technology, Institute of Chemical Technology, Prague
Nematode Caenorhabditis elegans could be a suitable model to study metabolic and cellular consequences of homocystinuria due to cystathionine β-synthase (CBS) deficiency. However, metabolism of sulfur amino acids in C. elegans is as yet unknown, namely the steps in cysteine biosynthetic pathways. Cysteine can be synthesized either via the transsulfuration pathway which utilizes homocysteine by CBS or via the assimilation pathway which uses sulfide by cysteine synthase (CS).
In silico analysis of C. elegans
database identified four homologs of human CBS, namely ZC373.1,
C17G1.7, K10H10.2 and R08E5.2. The aim of this study was to
express the gene C17G1.7 (predicted CS) in prokaryotic system, to purify
and further characterize this recombinant protein. Molecular weight of
polypeptide chain was determined to be 37,2 kDa by MALDI-TOF MS. Blue Native
electrophoresis revealed a molecular weight of 70 kDa suggesting that
recombinant CS is a dimer. Purified protein contains pyridoxal 5'-phosphate
(PLP) as determined by UV/VIS absorption spectrometry; circular dichroism
showed characteristic PLP maximum confirming its localization in a centre of
organized globular protein. We determined that purified enzyme has very
specific enzymic activity for CS reaction; other possible activities were not
detected. Recombinant CS exhibited Km values for O-acetyl-L-serine
and sulfide of 5.54 and
These data show that C17G1.7 could play an important role in cysteine biosynthesis since C. elegans genome contains also a CBS gene, we hypothesize that nematode utilizes both cysteine biosythesis pathways - sulfur assimilation and transsulfuration pathway.
This work was supported by Wellcome trust International senior research fellowship in biomedical science in central europe