The role of cysteine residues 129 and 329 in Escherichia coli K1 CMP-NeuAc synthase

Abstract

N-Acetylneuraminic acid cytidyltransferase (CMP-NeuAc synthase) of Escherichia coli K1 is sensitive to mercurials and has cysteine residues only at positions 129 and 329. The role of these residues in the catalytic activity and structure of the protein has been investigated by site-directed mutagenesis and chemical modification. The enzyme is inactivated by the thiol-specific reagent dithiodipyridine. Inactivation by this reagent is decreased in the presence of the nucleotide substrate CTP, suggesting that a thiol residue is at or near the active site. Site-directed mutagenesis of either residue Cys-129 to serine or Cys-329 to selected amino acids has minor effects on the specific activity of the enzyme, suggesting that cysteine is not essential for catalysis and that a disulphide bond is not an essential structural component. The limited reactivity of the enzyme to other thiol-blocking reagents suggests that its cysteine residues are partially exposed. The accessibility and role of the cysteine residues in enzyme structure were investigated by fluorescence, c.d. and denaturation studies of wild-type and mutant enzymes. The mutation of Cys-129 to serine makes the enzyme more sensitive to heat and chemical denaturation, but does not cause gross changes in the protein structure as judged by the c.d. spectrum. The mutant containing Ser-129 instead of Cys-129 had a complex denaturation pathway similar to that of wild-type E. coli K1 CMP-NeuAc synthase consisting of several partially denatured states. Cys-329 reacts more readily with N-[14C]ethylmaleimide when the enzyme is in a heat-induced relaxed state. Cys-129 is less reactive and is probably a buried residue.

Chemistry, biochemistry and biology of sialic acids

“…However, exchange of these cysteine residues for other amino acids by site-directed mutagenesis did not change the enzyme activity significantly [590]. In the recombinant enzyme from E. coli two cysteine residues were recognized at the positions 129 and 329, one of which seems to be at or close to the active center, as was shown by inhibition with thiol-specific reagents partly in the presence of CTP.…”

Separate Pathways for O Acetylation of Polymeric and Monomeric Sialic Acids and Identification of Sialyl O -Acetyl Esterase in Escherichia coli K1

“…Recombinant, histidine-tagged endo-N-acetylneuraminidase (endo-N), from phage PK1E, was purified from a culture of IPTG (isopropyl-␤-Dthiogalactoside)-induced E. coli BL21(DE3) essentially as described previously (30). CMP-Neu5Ac synthetase was purified as previously described (53,54) from a culture of induced cells expressing pWV200b and had a specific activity of 610.3 units/mg protein, where 1 unit activates 1 mol of Neu5Ac in 1 min at 37°C. Other enzymes were used as induced soluble extracts from cells (BL21 or DH5␣) grown in overexpression broth (Zymo Research, Orange, CA) with 1 mM IPTG.…”

“…The bacterial strains and plasmids used in this study are shown in Table 1. Plasmid pWV200b used for overproduction of E. coli K1 NeuA was derived from pWA1 (53,54) as a template using PCR primers containing EcoRI and HindIII sites. Double-digested PCR product was cloned into similarly digested pKK223-3, placing K1 neuA ϩ under control of the tac promoter and the ribosome binding site of pKK223-3.…”

lec32 Is a New Mutation in Chinese Hamster Ovary Cells That Essentially Abrogates CMP-N-acetylneuraminic Acid Synthetase Activity

“…Since both the E. coli (42) and rat liver (40) CMP-NeuAc synthetase enzymes are known to be sensitive to NEM, the CHO enzyme was tested. It was found that, whereas very low levels of NEM were slightly stimulatory, the addition of 1 mM NEM to the reaction mixture reduced the CHO activity to about 12%, while the activity of the purified E. coli enzyme was only reduced to 88% under these conditions (Fig.…”