Endo/exo mechanism and processivity of family 18 chitinases produced by Serratia marcescens.
We present a comparative study of ChiA, ChiB, and ChiC, the three family 18 chitinases produced by Serratia marcescens. All three enzymes eventually converted chitin to N-acetylglucosamine dimers (GlcNAc2) and a minor fraction of monomers. ChiC differed from ChiA and ChiB in that it initially produced longer oligosaccharides from chitin and had lower activity towards an oligomeric substrate, GlcNAc6. ChiA and ChiB could convert GlcNAc6 directly to three dimers, whereas ChiC produced equal amounts of tetramers and dimers, suggesting that the former two enzymes can act processively. Further insight was obtained by studying degradation of the soluble, partly deacetylated chitin-derivative chitosan. Because there exist nonproductive binding modes for this substrate, it was possible to discriminate between independent binding events and processive binding events. In reactions with ChiA and ChiB the polymer disappeared very slowly, while the initially produced oligomers almost exclusively had even-numbered chain lengths in the 2-12 range. This demonstrates a processive mode of action in which the substrate chain moves by two sugar units at a time, regardless of whether complexes formed along the way are productive. In contrast, reactions with ChiC showed rapid disappearance of the polymer and production of a continuum of odd- and even-numbered oligomers. These results are discussed in the light of recent literature data on directionality and synergistic effects of ChiA, ChiB and ChiC, leading to the conclusion that ChiA and ChiB are processive chitinases that degrade chitin chains in opposite directions, while ChiC is a nonprocessive endochitinase.
Horn
Svein J
SJ
Department of Chemistry, Biotechnology and Food Science, the Norwegian University of Life Sciences, As, Norway.
Sørbotten
Audun
A
Synstad
Bjørnar
B
Sikorski
Pawel
P
Sørlie
Morten
M
Vårum
Kjell M
KM
Eijsink
Vincent G H
VG
D002686:Chitin / Q000378:metabolism; D002688:Chitinases / Q000737:chemistry / Q000145:classification* / Q000378:metabolism*; D048271:Chitosan / Q000378:metabolism; D019281:Dimerization; D004789:Enzyme Activation; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics*; D013045:Species Specificity; D013997:Time Factors
D002686:Chitin; D048271:Chitosan; D002688:Chitinases