Detection and characterisation of the genes encoding glyoxalase I and II from Neisseria meningitidis

Abstract

Glyoxalase enzymes I and II are involved in a detoxi®cation process consisting of conversion of reactive dicarbonyl compounds (e.g., methylglyoxal) to less reactive hydroxy acids. The structural gene for meningococcal glyoxalase I ( gloA) was identi®ed by screening an expression library with a rabbit antiserum. The meningococcal gloA gene consisted of 138 deduced amino acids, with a calculated mol. wt of 15.7 kDa. The DNA and deduced protein sequence of gloA was compared to known sequences of glyoxalase I enzymes and showed high homology with gloA of several eukaryotic and prokaryotic species. Insertion of a gloA-containing plasmid in Escherichia coli increased the host organism's tolerance to methylglyoxal from <2 mM to >4 mM, thus demonstrating its functional identity. A databank search also revealed the presence of a putative gloB gene, encoding glyoxalase II (GlxII), in the recently released genomic sequences of Neisseria meningitidis and N. gonorrhoeae.

Resistance ofNeisseria meningitidisto the Toxic Effects of Heme Iron and Other Hydrophobic Agents Requires Expression ofght

“…Overlapping the 3Ј end of NMA2148 is gloA (Fig. 2), a glyoxalase I gene described previously (14). One additional transposon mutant (strain 15F) displayed an identical phenotype to 276D, which was subsequently determined to have a transposon insertion in NMA2148.…”

T-Cell-Stimulating Protein A Elicits Immune Responses during Meningococcal Carriage and Human Disease

“…Sequence analysis of tspA (EMBL accession number AJ010113) revealed that the 2,625-bp gene encodes a protein consisting of 875 amino acids (aa) with a predicted molecular mass of 92,443 Da, a pI of 4.13, and a net charge at pH 7.0 of Ϫ85.99. In the published genome sequence of strain MC58 (serogroup B; GenBank accession number AAF40784 [39]) the gene was flanked by the lactoylglutathione lyase gene (gloA; accession number AAF40783.1 [20]) and a homolog of the intracellular septation protein gene (ispA; accession number AAF40785.1). Both flanking genes are in the opposite orientation with respect to tspA, and a promoter is predicted upstream of tspA, indicating that it is monocistronic.…”

Biochemistry of the Nickel‐Dependent Glyoxalase I Enzymes

“…Putative GlxI sequences have also been identifi ed and reported, underlining the broad occurrence of the enzyme system in Nature [57][58][59][60][61][62][63][64][65][66][67][68]. A search (as of January 2006) of the National Center for Biotechnology Information (NCBI) Protein Database reveals a diverse distribution of unique putative GlxI sequences: 70 bacterial sequences, 27 from the metazoan class, 34 fungal sequences, 8 from plant sources and 10 other eukaryotic sequences mainly from Plasmodium spp [69].…”

“…However it has been the detailed studies of the GlxI enzymes from human, Saccharomyces cerevisiae, rat, and Pseudomonas putida that have yielded the most detailed information at the mechanistic level [72][73][74][75][76][77]. Recently, the cloning and expression of a number of other bacterial GlxI (E. coli, Pseudomonas aeruginosa, Neisseria meningitidis and Yersinia pestis) have contributed to additional and fascinating information about the GlxI enzymes [67,78,79]. …”

“…Based on sequence comparisons, several bacterial genomes were scanned for putative GlxI sequences using the E. coli GlxI enzyme as a query [59]. Putative GlxI-encoding genes from Y. pestis, P. aeruginosa and the previously identifi ed, but uncharacterized enzyme from N. meningitidis were cloned into E. coli cell lines for recombinant protein production ( Figure 6) [67,79]. All three enzymes were expressed abundantly and possessed GlxI activity.…”