Eukaryotic NAD+Synthetase Qns1 Contains an Essential, Obligate Intramolecular Thiol Glutamine Amidotransferase Domain Related to Nitrilase

ABSTRACT Streptococcus sobrinus, one agent of dental caries, secretes a protein that induces lymphocyte polyclonal activation of the host as a mechanism of immune evasion. We have isolated from culture supernatants of this bacterium a protein with murine B-cell-stimulatory properties and subsequently cloned the relevant gene. It contains an open reading frame of 825 bp encoding a polypeptide with 275 amino acid residues and a molecular mass of 30 kDa. The protein displays high sequence homology with NAD+ synthetases from several organisms, including a conserved fingerprint sequence (SGGXD) characteristic of ATP pyrophosphatases. The polypeptide was expressed in Escherichia coli as a hexahistidine-tagged protein and purified in an enzymatically active form. The recombinant NAD+ synthetase stimulates murine B cells after in vitro treatment of spleen cell cultures, as demonstrated by its ability to induce up-regulation of the expression of CD69, an early marker of lymphocyte activation. Stimulation with the recombinant NAD+ synthetase was also observed with other B-cell markers, such as CD19+, B220+, and CD21+. Cell proliferation follows the activation induced by the recombinant NAD+ synthetase.

Download Full-text

Characterization of LtsA from Rhodococcus erythropolis, an Enzyme with Glutamine Amidotransferase Activity

Journal of Bacteriology ◽

10.1128/jb.187.8.2582-2591.2005 ◽

2005 ◽

Vol 187 (8) ◽

pp. 2582-2591 ◽

Cited By ~ 32

Author(s):

Yasuo Mitani ◽

XianYing Meng ◽

Yoichi Kamagata ◽

Tomohiro Tamura

Keyword(s):

Cell Wall ◽

Culture Media ◽

Rhodococcus Erythropolis ◽

Mycolic Acid ◽

Site Directed Mutagenesis ◽

Host Cells ◽

Wall Structure ◽

Synthetase Activity ◽

Glutamine Amidotransferase ◽

Glutaminase Activity

ABSTRACT The nocardioform actinomycete Rhodococcus erythropolis has a characteristic cell wall structure. The cell wall is composed of arabinogalactan and mycolic acid and is highly resistant to the cell wall-lytic activity of lysozyme (muramidase). In order to improve the isolation of recombinant proteins from R. erythropolis host cells (N. Nakashima and T. Tamura, Biotechnol. Bioeng. 86:136-148, 2004), we isolated two mutants, L-65 and L-88, which are susceptible to lysozyme treatment. The lysozyme sensitivity of the mutants was complemented by expression of Corynebacterium glutamicum ltsA, which codes for an enzyme with glutamine amidotransferase activity that results from coupling of two reactions (a glutaminase activity and a synthetase activity). The lysozyme sensitivity of the mutants was also complemented by ltsA homologues from Bacillus subtilis and Mycobacterium tuberculosis, but the homologues from Streptomyces coelicolor and Escherichia coli did not complement the sensitivity. This result suggests that only certain LtsA homologues can confer lysozyme resistance. Wild-type recombinant LtsA from R. erythropolis showed glutaminase activity, but the LtsA enzymes from the L-88 and L-65 mutants displayed drastically reduced activity. Interestingly, an ltsA disruptant mutant, which expressed the mutated LtsA, changed from lysozyme sensitive to lysozyme resistant when NH4Cl was added into the culture media. The glutaminase activity of the LtsA mutants inactivated by site-directed mutagenesis was also restored by addition of NH4Cl, indicating that NH3 can be used as an amide donor molecule. Taken together, these results suggest that LtsA is critically involved in mediating lysozyme resistance in R. erythropolis cells.

Download Full-text