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Identification of the gonococcal glmU gene encoding the enzyme N-acetylglucosamine 1-phosphate uridyltransferase involved in the synthesis of UDP-GlcNAc.

ABSTRACT: In searching for the gonococcal sialyltransferase gene(s), we cloned a 3.8-kb DNA fragment from gonococcus strain MS11 that hybridized with the oligonucleotide JU07, which was derived from the conserved C terminus of the sialyl motif present in mammalian sialyltransferases. Sequencing of the fragment revealed four putative open reading frames (ORFs), one of which (ORF-1) contained a partial sialyl motif including the amino acid sequence VGSKT, which is highly conserved among sialyltransferases. The gene was flanked by two inverted repeats containing the neisserial DNA uptake sequence and was preceded by a putative sigma 54 promoter. Database searches, however, revealed a high degree of homology between ORF-1 and the N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) of Escherichia coli and Bacillus subtilis and not with any known sialyltransferase. This homology was further established by the successful complementation of an orf-1 mutation by the E. coli glmU gene. Enzyme assays demonstrated that ORF-1 did not possess sialyltransferase activity but mimicked GlmU function catalyzing the conversion of N-acetylglucosamine 1-phosphate into UDP-N-acetylglucosamine, which is a key metabolite in the syntheses of lipopolysaccharide, peptidoglycan, and sialic acids.

SUBMITTER: Ullrich J

PROVIDER: S-EPMC177559 | biostudies-other | 1995 Dec

REPOSITORIES: biostudies-other

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Identification of the gonococcal glmU gene encoding the enzyme N-acetylglucosamine 1-phosphate uridyltransferase involved in the synthesis of UDP-GlcNAc.

Ullrich J J van Putten J P JP

Journal of bacteriology 19951201 23

In searching for the gonococcal sialyltransferase gene(s), we cloned a 3.8-kb DNA fragment from gonococcus strain MS11 that hybridized with the oligonucleotide JU07, which was derived from the conserved C terminus of the sialyl motif present in mammalian sialyltransferases. Sequencing of the fragment revealed four putative open reading frames (ORFs), one of which (ORF-1) contained a partial sialyl motif including the amino acid sequence VGSKT, which is highly conserved among sialyltransferases. ...[more]

PMID: 7592484

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Project description:UDP-N-acetylglucosamine:dolichyl-phosphate N-acetylglucosamine-1-phosphate transferase (GPT) is the first enzyme in the dolichol pathway of protein N-glycosylation, and is implicated in the developmental programmes of a variety of eukaryotes. In the present study we describe the effects of all-trans-retinoic acid (RA) on the levels of GPT protein and enzymic activity, and on the transcription rate of the GPT gene, in mouse P19 teratocarcinoma cells. RA caused a dose-dependent and protein-synthesis-dependent induction of enzyme activity. The maximum induction of GPT activity (about 3-fold) required 2 days of exposure to 1 microM RA. Induced GPT activity also resulted in an increase in the rate of incorporation of [3H]mannose into Glc3Man9GlcNAc2. Enzymic activities paralleled GPT gene expression. The GPT gene was induced (2-fold) after 7 h of RA treatment. An approx. 3-fold increase in a 48 kDa GPT protein and approx. 4-fold increases in the levels of three GPT transcripts (1.8, 2.0 and 2.2 kb) were observed after 2 days of RA treatment. The enhanced levels of GPT protein and mRNAs began to decline 3 days after the initiation of differentiation, and GPT expression was down-regulated during cellular differentiation. GPT activity decreased about 2. 8-fold to a constant level in differentiated P19 cells. The results indicate that the RA-induced enzyme activity was mainly determined by increased transcription of the GPT gene. RA-treated P19 cells were about 4-fold more resistant to tunicamycin, a fungal antibiotic which inhibits GPT, than were control cells. In addition, GPT activity in membranes from RA-treated P19 cells exhibited approx. 4-fold increased resistance to tunicamycin compared with activity in membranes from untreated control cells, demonstrating that resistance to tunicamycin is correlated with induced GPT activity. Furthermore, increased GPT activity had regulatory significance with regard to the rate of incorporation of [3H]mannose into Glc3Man9GlcNAc2-P-P-dolichol and into glycoproteins. Together, the data provide additional insights into the hormonal regulation of GPT and present evidence that the RA-mediated induction of GPT has a regulatory impact on the dolichol pathway.

Dataset Information

Identification of the gonococcal glmU gene encoding the enzyme N-acetylglucosamine 1-phosphate uridyltransferase involved in the synthesis of UDP-GlcNAc.

Publications

Identification of the gonococcal glmU gene encoding the enzyme N-acetylglucosamine 1-phosphate uridyltransferase involved in the synthesis of UDP-GlcNAc.

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