Some studies on catalysis by lysozyme

Ballardie, F. W.; Capon, Brian; Cuthbert, Murray W.; Dearie, William M.

doi:10.1016/0045-2068(77)90047-5

Cited by 26 publications

(5 citation statements)

References 45 publications

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“…The XylNac residue, however, was used as a chemical substitution for the GlcNAc residue in peptidoglycan for the study of the polysaccharide hydrolytic enzyme, lysozyme (34). Interestingly, the B. thuringiensis peptidoglycan cell wall is recalcitrant to hydrolysis by lysozyme (35).…”

Section: Discussionmentioning

confidence: 99%

Biosynthesis of a New UDP-sugar, UDP-2-acetamido-2-deoxyxylose, in the Human Pathogen Bacillus cereus Subspecies cytotoxis NVH 391-98

Glushka

Lee

et al. 2010

Journal of Biological Chemistry

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We have identified an operon and characterized the functions of two genes from the severe food-poisoning bacterium, Bacillus cereus subsp. cytotoxis NVH 391-98, that are involved in the synthesis of a unique UDP-sugar, UDP-2-acetamido-2-deoxyxylose (UDP-N-acetyl-xylosamine, UDP-XylNAc). UGlcNAcDH encodes a UDP-N-acetyl-glucosamine 6-dehydrogenase, converting UDP-N-acetylglucosamine (UDP-GlcNAc) to UDP-N-acetyl-glucosaminuronic acid (UDP-GlcNAcA). The second gene in the operon, UXNAcS, encodes a distinct decarboxylase not previously described in the literature, which catalyzes the formation of UDPXylNAc from UDP-GlcNAcA in the presence of exogenous NAD ؉ . UXNAcS is specific and cannot utilize UDP-glucuronic acid and UDP-galacturonic acid as substrates. UXNAcS is active as a dimer with catalytic efficiency of 7 mM ؊1 s ؊1. The activity of UXNAcS is completely abolished by NADH but unaffected by UDP-xylose. A real-time NMR-based assay showed unambiguously the dual enzymatic conversions of UDP-GlcNAc to UDP-GlcNAcA and subsequently to UDP-XylNAc. From the analyses of all publicly available sequenced genomes, it appears that UXNAcS is restricted to pathogenic Bacillus species, including Bacillus anthracis and Bacillus thuringiensis. The identification of UXNAcS provides insight into the formation of UDP-XylNAc. Understanding the metabolic pathways involved in the utilization of this amino-sugar may allow the development of drugs to combat and eradicate the disease.A food poisoning outbreak in France in 1998 led to the isolation of a new strain of Bacillus cereus subsp. cytotoxis NVH 391-98 (1). This rod-shaped Gram-positive bacterium causes a disease that initially produces emetic (nausea and vomiting)-like symptoms, and/or in more severe cases produces a diarrheal form that causes abdominal cramps and diarrhea (2). Similar to its close relatives, the notorious human pathogen Bacillus anthracis and the insecticidal Bacillus thuringiensis, the B. cereus strain can form spores. Due to its cell surface, a spore can survive harsh conditions (e.g. soil and air), and when the environment becomes appropriate, it will germinate, resulting in a vegetative cell that can produce emetic toxin and different enterotoxins (3). The cell surfaces of many pathogenic bacteria are composed of diverse and complex carbohydrate structures, some of which are known virulence factors. Indeed, different B. cereus peptidoglycans and glycoproteins were isolated, and some were reported to play a role in spore formation and infection (3-5). It is also clear, however, that among those different types of Bacillus glycans, many are not yet fully characterized. Regardless, compared with the limited knowledge of these glycan structures, the pathways leading to their biosyntheses are still elusive. To identify such metabolic pathways, we initially decided to look for putative genes encoding enzymes involved in the synthesis of glycan precursors (i.e. nucleotide-sugars).Different UDP-GlcA 2 decarboxylases with distinct functions exist in both eukaryotes...

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Section: Discussionmentioning

confidence: 99%

Biosynthesis of a New UDP-sugar, UDP-2-acetamido-2-deoxyxylose, in the Human Pathogen Bacillus cereus Subspecies cytotoxis NVH 391-98

Glushka

Lee

et al. 2010

Journal of Biological Chemistry

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“…Mutation of the catalytic acid/base residue of HEWL and other bretaining glycosidases has been shown to greatly reduce the rate of hydrolysis of unactivated glycosides by slowing both steps of the reaction 11,12 . Substrates bearing activated leaving groups do not require signi®cant acid catalytic assistance and so react at near wildtype rates to yield the glycosyl-enzyme intermediate 12 chitobiosyl¯uoride (NAG 2 F) 13 we observed, using electrospray ionization mass spectrometry (ESI-MS), a mixture of the free enzyme HEWL(E35Q) and a high steady-state population (Fig. 2b) To reduce the rate of the second step in a different approach, we incorporated¯uorine in place of the 2-acetamido group of chitobiose, while keeping a good leaving group at the anomeric centre.…”

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confidence: 99%

Catalysis by hen egg-white lysozyme proceeds via a covalent intermediate

Vocadlo

Davies

Laine

et al. 2001

Nature

596

456

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Hen egg-white lysozyme (HEWL) was the first enzyme to have its three-dimensional structure determined by X-ray diffraction techniques. A catalytic mechanism, featuring a long-lived oxocarbenium-ion intermediate, was proposed on the basis of model-building studies. The 'Phillips' mechanism is widely held as the paradigm for the catalytic mechanism of beta-glycosidases that cleave glycosidic linkages with net retention of configuration of the anomeric centre. Studies with other retaining beta-glycosidases, however, provide strong evidence pointing to a common mechanism for these enzymes that involves a covalent glycosyl-enzyme intermediate, as previously postulated. Here we show, in three different cases using electrospray ionization mass spectrometry, a catalytically competent covalent glycosyl-enzyme intermediate during the catalytic cycle of HEWL. We also show the three-dimensional structure of this intermediate as determined by X-ray diffraction. We formulate a general catalytic mechanism for all retaining beta-glycosidases that includes substrate distortion, formation of a covalent intermediate, and the electrophilic migration of C1 along the reaction coordinate.

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“…However, this method is not always reproducible because of the dependence on the ionic strength of the medium 47,48 . Different methods using small synthetic substrates have been developed, investigated and recommended for accurate determination of LS [49][50][51] . Observation of the bioactive fraction of a-CH released from microparticles prepared using PGA-co-PDL and PPA-co-PDL ( Figure 6) indicates that the maximum bioactivity was observed at zero hours and ranged between 27% and 60%.…”

Section: Enzyme Bioactivitymentioning

confidence: 99%