Saccharomyces cerevisiae chitin biosynthesis activation by N-acetylchitooses depends on size and structure of chito-oligosaccharides.

BACKGROUND: To explore chitin synthesis initiation, the effect of addition of exogenous oligosaccharides on in vitro chitin synthesis was studied. Oligosaccharides of various natures and lengths were added to a chitin synthase assay performed on a Saccharomyces cerevisiae membrane fraction. FINDINGS: N-acetylchito-tetra, -penta and -octaoses resulted in 11 to 25% [14C]-GlcNAc incorporation into [14C]-chitin, corresponding to an increase in the initial velocity. The activation appeared specific to N-acetylchitooses as it was not observed with oligosaccharides in other series, such as beta-(1,4), beta-(1,3) or alpha-(1,6) glucooligosaccharides. CONCLUSIONS: The effect induced by the N-acetylchitooses was a saturable phenomenon and did not interfere with free GlcNAc and trypsin which are two known activators of yeast chitin synthase activity in vitro. The magnitude of the activation was dependent on both oligosaccharide concentration and oligosaccharide size.

Expression in E. coli and characterization of the catalytic domain of Botrytis cinerea chitin synthase.

BACKGROUND: Chitin synthase 3a (CHS3a) from Botrytis cinerea (Bc) catalyses the multiple transfer of N-acetylglucosamine (GlcNAc) residues to the growing chitin chain. Chitin, a ß-1,4 linked GlcNAc homopolymer, is an essential cell wall component of filamentous fungi. Chitin synthase, processive membranous protein, has been recognized as a promising target for new antifungicides. Enzymatic characterizations of chitin synthases have been limited, mainly because purity and amounts of integral enzyme obtained after purification procedures have not been sufficient. FINDINGS: We undertook the preparation of two BcCHS3a fragment proteins, containing only the central domain and devoid of the N-terminal and transmembrane C-terminal regions. The central domain of CHS3a, named SGC (Spsa GntI Core), is conserved in all UDP-glycosyltransferases and it is believed to contain the active site of the enzyme. CHS3a-SGC protein was totally expressed as inclusion bodies in Escherichia coli. We performed recombinant CHS3a-SGC purification in denaturing conditions, followed by a refolding step. Although circular dichroism spectra clearly exhibited secondary structures of renatured CHS3a-SGC, no chitin synthase activity was detected. Nevertheless CHS3a-SGC proteins show specific binding for the substrate UDP-GlcNAc with a dissociation constant similar to the Michaelis constant and a major contribution of the uracil moiety for recognition was confirmed. CONCLUSIONS: Milligram-scale quantities of CHS3a-SGC protein with native-like properties such as specific substrate UDP-GlcNAc binding could be easily obtained. These results are encouraging for subsequent heterologous expression of full-length CHS3a.

Chemoenzymatic synthesis of stable isotope labeled UDP-N-[2H]-acetyl-glucosamine and [2H]-acetyl-chitooligosaccharides.

Labeled UDP-GlcNAc and chitooligosaccharides should be powerful tools for studies of N-acetylglucosaminyltransferase such as chitin synthases. We describe here a rapid, inexpensive and a common strategie for the chemoenzymatic synthesis of uridine 5'-diphospho-N-[(2)H]-acetyl-glucosamine and the chemical preparation of N-[(2)H]-acetyl chitooligosaccharides (from 2 to 5 mers). Deuterated UDP-GlcNAc analogue was tested as chitin synthase substrate and it exhibited an incorporation level in chitin as the natural substrate. Deuterium labeling of carbohydrates present different advantages: it is a stable isotope and allows glycosyltransferase mechanism studies by a mass spectrometry approach.

Synthesis of protected 2-amino-2-deoxy-D-xylothionolactam derivatives and some aspects of their reactivity.

The synthesis of polyfunctionalized delta-lactams as key intermediates of glycomimetics in the 2-acetamido-2-deoxy sugar series is presented. Starting from a chiral gamma-amino vinylic ester synthesized from Garner's aldehyde and after regioselective reduction, 1-azido-3-(N-tert-butyloxycarbonyl-2,2-dimethyloxazolidin-4-yl)-2-propene was obtained. Next, a cis-dihydroxylation reaction provided the protected D-xylitol and L-arabinitol azides. A simple protection-deprotection sequence, followed by an oxidation and a reductive cyclization, led to protected 2-amino-delta-lactams bearing a tert-butyloxycarbonyl group on the amine functionality. To explore the reactivity of such compounds, activation of the lactam into the corresponding thionolactam was performed. The resulting 2-amino-D-xylothionolactam derivative, a versatile intermediate, allowed access to a first generation of protected 2-amino-D-xylosamidoxime derivatives which are of interest as precursors of N-acetylhexosaminidase and N-acetylglucosaminyltransferase inhibitors. In this series of compounds, epimerization at C-2 was observed. AM(1) calculations performed on these analogs showed that they adopted a B(2,5) conformation and that the axial epimer was favored in the protected series whereas the equatorial epimer was preferred in the unprotected series.

Synthesis of new 3-alkoxy-7-amino-4-chloro-isocoumarin derivatives as new beta-amyloid peptide production inhibitors and their activities on various classes of protease.

A series of new 7-substituted-4-chloro-3-alkoxy isocoumarin derivatives were synthesized and evaluated as inhibitors of representative classes of proteases: serine protease (alpha-chymotrypsin, trypsin), cysteine protease (Caspase-3), and aspartyl protease (HIV-protease), 20S proteasome and also as inhibitors of amyloid peptide gamma-secretase-mediated production. Protease inhibition selectivity is directly related to the structure of the substituent at the 7-position of the isocoumarin nucleus. 7-Nitro-isocoumarin derivatives (4c, 4d, 4f) are potent alpha-chymotrypsin inhibitors but slightly active or inactive on HIV-protease, as well as on cysteine protease. In contrast, only derivatives bearing a free amino (5d, 5f) or a substituted amino group (6f) at the 7-position of the isocoumarin nucleus, were found weakly active or inactive on alpha-chymotrypsin, trypsin, Caspase-3 and HIV-protease, but prevent gamma-secretase-mediated production of Abeta 40/42 amyloid peptides, which is known to be involved in Alzheimer's disease. Moreover, the most active compounds on beta-amyloid peptide production [JLK6 (5d), JLK2 (5f) and JLK7 (6f)] show only weak or moderate inhibitory activity on the 20S proteasome. The obtained results suggest that the described new isocoumarin analogues could be of interest, since compounds like JLK6 (5d), JLK2 (5f) and JLK7 (6f) can be considered as possible hits for the development of new agents directed towards Alzheimer's disease.