The chitinolytic system of Lactococcus lactis ssp. lactis comprises a nonprocessive chitinase and a chitin-binding protein that promotes the degradation of alpha- and beta-chitin.
It has recently been shown that the Gram-negative bacterium Serratia marcescens produces an accessory nonhydrolytic chitin-binding protein that acts in synergy with chitinases. This provided the first example of the production of dedicated helper proteins for the turnover of recalcitrant polysaccharides. Chitin-binding proteins belong to family 33 of the carbohydrate-binding modules, and genes putatively encoding these proteins occur in many microorganisms. To obtain an impression of the functional conservation of these proteins, we studied the chitinolytic system of the Gram-positive Lactococcus lactis ssp. lactis IL1403. The genome of this lactic acid bacterium harbours a simple chitinolytic machinery, consisting of one family 18 chitinase (named LlChi18A), one family 33 chitin-binding protein (named LlCBP33A) and one family 20 N-acetylhexosaminidase. We cloned, overexpressed and characterized LlChi18A and LlCBP33A. Sequence alignments and structural modelling indicated that LlChi18A has a shallow substrate-binding groove characteristic of nonprocessive endochitinases. Enzymology showed that LlChi18A was able to hydrolyse both chitin oligomers and artificial substrates, with no sign of processivity. Although the chitin-binding protein from S. marcescens only bound to beta-chitin, LlCBP33A was found to bind to both alpha- and beta-chitin. LlCBP33A increased the hydrolytic efficiency of LlChi18A to both alpha- and beta-chitin. These results show the general importance of chitin-binding proteins in chitin turnover, and provide the first example of a family 33 chitin-binding protein that increases chitinase efficiency towards alpha-chitin.
Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, As, Norway. gustav.vaaje-kolstad@umb.no
Gustav
G
Vaaje-Kolstad
Anne C
AC
Bunaes
Geir
G
Mathiesen
Vincent G H
VG
Eijsink
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D002686:Chitin / Q000737:chemistry / Q000378:metabolism*; D002688:Chitinases / Q000737:chemistry* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012706:Serratia marcescens / Q000201:enzymology / Q000378:metabolism
D001426:Bacterial Proteins; D002686:Chitin; D004247:DNA; D002688:Chitinases