Comparative studies of chitinases A, B and C fromSerratia marcescens

Abstract

Serratia marcescens produces three chitinases, ChiA, ChiB and ChiC which together enable the bacterium to efficiently degrade the insoluble chitin polymer. We present an overview of the structural properties of these enzymes, as well as an analysis of their activities towards artificial chromogenic chito-oligosaccharide-based substrates, chito-oligosaccharides, chitin and chitosan. We also present comparative inhibition data for the pseudotrisaccharide allosamidin (an analogue of the reaction intermediate) and the cyclic pentapeptide argadin. The results show that the enzymes differ in terms of their subsite architecture and their efficiency towards chitinous substrates. The idea that the three chitinases play different roles during chitin degradation was confirmed by the synergistic effects that were observed for certain combinations of the enzymes. Studies of the degradation of the soluble heteropolymer chitosan provided insight into processivity. Taken together, the available data for Serratia chitinases show that the chitinolytic machinery of this bacterium consists of two processive exoenzymes that degrade the chitin chains in opposite directions (ChiA and ChiB) and a non-processive endo-enzyme, ChiC.

Determination of substrate binding energies in individual subsites of a family 18 chitinase

“…Thus, some degree of substrate affinity may be expected in additional aglycon subsites, and this has indeed been observed [25]. Previous studies of ChiB catalyzed hydrolysis of CHOS have shown that (GlcNAc) 3 productively binds from À2 to +1, (GlcNAc) 4 productively binds from À2 to +2, and that (GlcNAc) 5 productively binds from À2 to +3 [25]. The preferential binding of (GlcNAc) 5 to subsites À2 to +3 was confirmed by the crystal structure of this ligand in complex with ChiB-E144Q [26].…”

“…The preferential binding of (GlcNAc) 5 to subsites À2 to +3 was confirmed by the crystal structure of this ligand in complex with ChiB-E144Q [26]. (GlcNAc) 6 productively binds from À3 to +3 (approximately 20 %) and from À2 to ''+4" (80%; ''+4" denotes a binding position next to the +3 subsite) [25]. Combining this information with the obtained thermodynamic parameters (Table 1) allows for the estimation of binding energies for individual subsites.…”

“…The binding affinity of this subsite is comparable to the binding affinity generated by a similar Trp-GlcNAc stacking Table 1, combined with existing knowledge concerning the binding preferences of the various ligands; see text for details. Since (GlcNAc) 6 approximately binds 20% from À3 to +3 and 80% from À2 to ''+4" [25], the difference in free energy change of À0.9 kcal/mol between (GlcNAc) 6 and (GlcNAc) 5 can be shared as À0.3 kcal/mol and À1.1 kcal/mol for the À3 and ''+4" subsites, respectively (solved as two equations with two unknowns, X, the DG for subsite À3 and Y, the DG for subsite +4; the equations are 0.2X + 0.8Y = 0.9 and (referring the experimentally observed binding ratios) Y = 4X). Estimates for DH and DS for these subsites cannot be made.…”

Chitinase inhibitors: extraction of the active framework from natural argifin and use of in situ click chemistry

“…FTD-0668), and was found to be a potent inhibitor of blowfly 6 and Serratia marcescens chitinases (SmChi). [9][10][11][12] Recently, argifin complexes with fungal (Aspergillus fumigatus), human and bacterial chitinases have been resolved by X-ray crystallography. 11,12 These studies revealed that there are at least four conserved hydrogenbond interactions between the N o -methylcarbamoyl-L-arginine moiety and the polar groups arrayed in the hydrolytic pocket of the family 18 chitinases examined to date.…”

Solid-phase total synthesis of the chitinase inhibitor Argadin using a supported acetal resin

“…FO-7314 and Gliocladium sp. FTD-0668, respectively, by our research group, and found to be potent chitinase inhibitors of blowfly (Lucilia cuprina) 10 and Serratia marcescens chitinases (SmChi), [13][14][15][16] both of which are family-18 chitinases (Figure 1). Developments of practical and efficient total syntheses of 1 and 2 are important objectives, as the original sources do not produce these cyclic peptides with sufficient quantities.…”