Applicant's Name_Orlean, Peter A.B.__Principle Investigator: Peter Orlean
Grant Title: How do chitin synthases start chitin chains and at which end do chains grow?
(a) Abstract:
1. Objectives. Hypotheses for how the enzymatic synthesis of chitin is initiated by
Glycosyltransferase Family 2 chitin synthases (CSs) were tested. These included thepossibility that CSs generate UDP-di-N-acetyl-chitobiose (UDP-GlcNAc2) and that UDPlinked chitin oligosaccharides (COs) generated by CSs could be trapped using a chainterminating UDP-GlcNAc analogue. The nature of the products of incubations waspredicted to provide evidence for (1) a discrete chitin initiation reaction between two UDPGlcNAcs and (2) for reducing- or non-reducing end chain extension. Additional analyses ofpriming of CO synthesis by a zymogenic CS (S. cerevisiae Chs1), and of the effects of a 2acylamido analogue of GlcNAc on fungal morphogenesis, were conducted.
2. Methods used. Saccharomyces cerevisiae chitin synthases 1 and 2 (Chs1 and Chs2) wereused as model CSs. They were overexpressed using a high copy vector under the control ofa strong inducible promoter in a yeast host strain lacking detectable background CSactivity. Membranes from these CS-overexpressing strains, which served as “in vivo
proteoliposomes”, were used as source of CS. Formation of both low- and high molecularweight reaction products (COs and insoluble chitin, respectively) was monitored using twotypes of assay. In the first, UDP-[14C]GlcNAc was used a substrate, and the effects ofprimers and the UDP-GlcNAc analogues UDP-GlcNAc2 and UDP-4-O-MeGlcNAc weretested. In the second, CO and chitin synthesis was assessed using a novel assay in which[14C]GlcNAc is used as priming substrate in the presence of unlabeled UDP-GlcNAc,
UDP-GlcNAc2 or UDP-4-O-MeGlcNAc. Proteolytically treated and untreated zymogenic
Chs1 was compared with Chs2 with respect to priming of CO formation by GlcNAc,
GlcNAc2 and the 2-acylamido derivatives of GlcN, GlcNPr, GlcNBu, and GlcNGc.
3. Results. Major findings are as follows.
i) We devised a [14C]GlcNAc priming assay that represents a new way to characterize CSs
that is distinct from UDP-GlcNAc-dependent chitin extension assays.
ii) UDP-GlcNAc2 is not a substrate for Chs1 or Chs2, so reducing end extension is unlikely.
iii) UDP-4-O-MeGlcNAc is neither used as substrate by Chs2, nor does it inhibit chitin orCO synthesis by Chs1 or Chs2. The UDP-GlcNAc-binding site of CSs therefore cannot
accommodate a CH3 group at the 4-OH of GlcNAc.
iv) Chs1 and Chs2 differ from one another in their oligosaccharide product profiles and intheir ability to use GlcNAc2 as primer. Such differences may impact in vivo CS function.
v) Partial proteolysis of Chs1 does not alter the processivity of this CS by converting a CO
synthase into a polysaccharide synthase, but does alter the response of this CS to GlcNAc.
vi) The 2-acylamido analogue of GlcNAc, GlcNBu, which primes CO synthesis by Chs1and Chs2, is taken up by the human pathogenic fungus Candida albicans and dramaticallyaffects cellular morphology in a way distinct from GlcNAc itself, potentially due to its
conversion to UDP-GlcNBu, and formation of GlcNBu-containing chitin analogue.