Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.5.1.4 (deaminase)
 5,113 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Peptidoglycan (PG) turnover in exponentially growing Neisseria gonorrhoeae RD5 type 4 was accompanied by release of soluble PG fragments into the medium. Turnover of the D-[14C]glucosamine-labeled glycan moiety and of the meso-[3H]diaminopimelic acid (DAP)-labeled peptide region occurred at similar rates (ca. 35% per generation). Turnover of D-[14C]alanine-labeled sites within the peptide side chain of PG occurred at roughly twice this rate; no turnover of L-[3H]proline-labeled protein was detected. Gel filtration of supernatants of cultures grown in the presence of labeled DAP, glucosamine, and D-alanine as described above and paper chromatography of hydrolyzed peak fractions revealed four major types of soluble PG. Two of these contained both peptide and glycan moieties and appeared to represent forms of disaccharide peptide monomers and dimers. The other two were (i) a 3H-labeled product lacking 14C and (ii) a 14C-containing product lacking 3H, which were similar in size to that expected for free tetrapeptides and free disaccharides, respectively. Together the appearance of these PG fragments and the concurrent turnover of glycan and peptide regions indicate that both glycan splitting and amidase PG hydrolase activities are involved in the turnover of PG in growing gonococci. If released during gonococcal infections, similar soluble PG fragments might influence the consequences of host-gonococcus interactions.
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PMID:Release of soluble peptidoglycan from growing gonococci: hexaminidase and amidase activities. 11 60

Two naturally occurring forms of gonococcal peptidoglycan (PG) were tested for their susceptibility to human PG hydrolases. Purified 3H-labeled PG substituted extensively with O-acetyl derivatives (O-PG; from Neisseria gonorrhoeae FA19) and 14C-labeled O-acetyl-deficient PG (non-O-PG; from N. gonorrhoeae RD5) were mixed together and treated with either normal human sera (NHS) or with lysozyme purified from human polymorphonuclear leukocytes (PMN-LZ). The initial rate of hydrolysis of O-PG by NHS or by PMN-LZ was two- to fourfold less than that of its non-O-PG counterpart in the same tube. When the reactions were allowed to go to completion. NHS solubilized both PGs completely, whereas PMN-LZ solubilized all of the non-O-PG and left ca. 60% of the O-PG insoluble. The PMN-LZ-soluble fraction of O-PG consisted largely of glycosidically linked fragments with molecular weights greater than ca. 10(4), whereas the corresponding non-O-PG was degraded to lower-molecular-weight fragments, exclusively. At completion, NHS hydrolyzed both PGs to fragments whose size was equal to or smaller than that of the free disaccharide unit of PG, suggesting that human sera contain a peptide-splitting (amidase) activity and a glycosidase activity, in addition to that of the well-known muramidase. NHS also promoted the release of high-molecular-weight PG fragments from intact gonococci. The persistence of human hydrolase-resistant PG in the form of soluble macromolecular fragments may potentiate the biological effects of gonococcal PG in vivo.
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PMID:Resistance of O-acetylated gonococcal peptidoglycan to human peptidoglycan-degrading enzymes. 640 67

Previous analysis of soluble peptidoglycan (PG) fragments released by exponentially growing gonococci implicated the combined action of both hexosaminidase and amidase activities in PG turnover. Current studies further characterized PG fragments which were labeled in the glycan with D-glucosamine and in the peptide moiety with meso-diaminopimelic acid of L- and D-alanine. Labeled PG fragments were isolated by gel filtration and characterized on the bases of (i) KD values, (ii) free amino group analysis using fluorodinitrobenzene, (iii) borohydride reduction, (iv) alkali-catalyzed beta-elimination, (v) paper chromatography in various solvents, (vi) electrophoretic mobility at various pH values, (vii) digestibility by Charonia lampas glycosidases, and (viii) content of labeled D- and L-alanine. A set of well-characterized PG fragments was used as standards. The monomer fraction (the major extracellular product) was found to contain two components. Most (about 80%) appeared to be N-acetylglucosaminyl-beta-1 leads to 4-1,6-anhydro-N-acetylmuramyl-L-ala-D-glu-meso-diaminopimelic acid; the remainder was the corresponding disaccharide tetrapeptide containing a C-terminal D-alanine. An unusual feature of these products was the presence of the anhydro-muramyl (non-reducing) ends, reflecting the activity of a gonococcal transglycosylase, and the near absence of products containing detectable reducing ends. Otherwise, the structures of the monomer fragments were typical of those expected for a gram-negative bacterium (chemotype I). The corresponding peptide-cross-linked dimer and the free disaccharide also contained nonreducing ends, exclusively. Free peptides (products of amidase activity) consisted of both tripeptide and tetrapeptide. In summary, all gonococci examined appear to possess an unusual transglycosylase activity which contributes to the release of soluble PG fragments containing nonreducing, anhydro-muramyl ends. The release of these fragments in vivo might be a unique aspect of gonococci-host interactions.
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PMID:Release of soluble peptidoglycan from growing conococci: demonstration of anhydro-muramyl-containing fragments. 677 63

Neisseria gonorrhoeae is prone to undergo autolysis under many conditions not conducive to growth. The role of autolysis during gonococcal infection is not known, but possible advantages for the bacterial population include provision of nutrients to a starving population, modulation of the host immune response by released cell components, and donation of DNA for natural transformation. Biochemical studies indicated that an N-acetylmuramyl-l-alanine amidase is responsible for cell wall breakdown during autolysis. In order to better understand autolysis and in hopes of creating a nonautolytic mutant, we mutated amiC, the gene for a putative peptidoglycan-degrading amidase in N. gonorrhoeae. Characterization of peptidoglycan fragments released during growth showed that an amiC mutant did not produce free disaccharide, consistent with a role for AmiC as an N-acetylmuramyl-l-alanine amidase. Compared to the wild-type parent, the mutant exhibited altered growth characteristics, including slowed exponential-phase growth, increased turbidity in stationary phase, and increased colony opacity. Thin-section electron micrographs showed that mutant cells did not fully separate but grew as clumps. Complementation of the amiC deletion mutant with wild-type amiC restored wild-type growth characteristics and transparent colony morphology. Overexpression of amiC resulted in increased cell lysis, supporting AmiC's purported function as a gonococcal autolysin. However, amiC mutants still underwent autolysis in stationary phase, indicating that other gonococcal enzymes are also involved in this process.
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PMID:AmiC functions as an N-acetylmuramyl-l-alanine amidase necessary for cell separation and can promote autolysis in Neisseria gonorrhoeae. 1701 60

The human-restricted pathogen Neisseria gonorrhoeae encodes a single N-acetylmuramyl-l-alanine amidase involved in cell separation (AmiC), as compared with three largely redundant cell separation amidases found in Escherichia coli (AmiA, AmiB, and AmiC). Deletion of amiC from N. gonorrhoeae results in severely impaired cell separation and altered peptidoglycan (PG) fragment release, but little else is known about how AmiC functions in gonococci. Here, we demonstrated that gonococcal AmiC can act on macromolecular PG to liberate cross-linked and non-cross-linked peptides indicative of amidase activity, and we provided the first evidence that a cell separation amidase can utilize a small synthetic PG fragment as substrate (GlcNAc-MurNAc(pentapeptide)-GlcNAc-MurNAc(pentapeptide)). An investigation of two residues in the active site of AmiC revealed that Glu-229 is critical for both normal cell separation and the release of PG fragments by gonococci during growth. In contrast, Gln-316 has an autoinhibitory role, and its mutation to lysine resulted in an AmiC with increased enzymatic activity on macromolecular PG and on the synthetic PG derivative. Curiously, the same Q316K mutation that increased AmiC activity also resulted in cell separation and PG fragment release defects, indicating that activation state is not the only factor determining normal AmiC activity. In addition to displaying high basal activity on PG, gonococcal AmiC can utilize metal ions other than the zinc cofactor typically used by cell separation amidases, potentially protecting its ability to function in zinc-limiting environments. Thus gonococcal AmiC has distinct differences from related enzymes, and these studies revealed parameters for how AmiC functions in cell separation and PG fragment release.
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PMID:Amidase Activity of AmiC Controls Cell Separation and Stem Peptide Release and Is Enhanced by NlpD in Neisseria gonorrhoeae. 2698 7

Neisseria gonorrhoeae releases peptidoglycan fragments during growth, and these molecules induce an inflammatory response in the human host. The proinflammatory molecules include peptidoglycan monomers, peptidoglycan dimers, and free peptides. These molecules can be released by the actions of lytic transglycosylases or an amidase. However, >40% of the gonococcal cell wall is cross-linked, where the peptide stem on one peptidoglycan strand is linked to the peptide stem on a neighboring strand, suggesting that endopeptidases may be required for the release of many peptidoglycan fragments. Therefore, we characterized mutants with individual or combined mutations in genes for the low-molecular-mass penicillin-binding proteins PBP3 and PBP4. Mutations in either dacB, encoding PBP3, or pbpG, encoding PBP4, did not significantly reduce the release of peptidoglycan monomers or free peptides. A mutation in dacB caused the appearance of a larger-sized peptidoglycan monomer, the pentapeptide monomer, and an increased release of peptidoglycan dimers, suggesting the involvement of this enzyme in both the removal of C-terminal d-Ala residues from stem peptides and the cleavage of cross-linked peptidoglycan. Mutation of both dacB and pbpG eliminated the release of tripeptide-containing peptidoglycan fragments concomitantly with the appearance of pentapeptide and dipeptide peptidoglycan fragments and higher-molecular-weight peptidoglycan dimers. In accord with the loss of tripeptide peptidoglycan fragments, the level of human NOD1 activation by the dacB pbpG mutants was significantly lower than that by the wild type. We conclude that PBP3 and PBP4 overlap in function for cross-link cleavage and that these endopeptidases act in the normal release of peptidoglycan fragments during growth.
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PMID:Neisseria gonorrhoeae PBP3 and PBP4 Facilitate NOD1 Agonist Peptidoglycan Fragment Release and Survival in Stationary Phase. 3051 Jan