Metabolism of cyclodextrins by Klebsiella oxytoca m5a1: purification and characterisation of a cytoplasmically located cyclodextrinase.
It has been shown previously that the products of 11 genes are required for metabolism of starch by Klebsiella oxytoca via a novel pathway. An extracellular cyclodextrin glucanotransferase first degrades starch into alpha- and beta-cyclodextrins; evidence then has been presented that the cyclodextrins are transported into the cytoplasma via a specific system and that they are metabolised inside the cell. To provide support for this model, we have analysed whether Klebsiella oxytoca possesses a cytoplasmic enzyme able to linearise cyclodextrins. A possible candidate was the product of the cymH gene since it displays sequence similarity with cyclodextrinases from other organisms. The cymH gene was overexpressed, and the CymH protein was purified. CymH is a monomer of 69 kDa molecular mass and hydrolysed cyclodextrins at an optimum pH of 7.0 and an optimum temperature of 23 degrees C, respectively. The apparent Km increased with increasing size of the cyclodextrins, but the reaction velocity decreased. Linear malto-oligosaccharides were also accepted as substrates, but were hydrolysed with a lower efficiency. Final products in each case were maltose and maltotriose. It was demonstrated by immunoblotting that CymH is located in the cytoplasm and that no signal peptide was cleaved off. Since cymH mutants were no longer able to grow on cyclodextrins, these results prove that cyclodextrins are degraded inside the cell, and they support the contention of the existence of a specific transport system.
Feederle
R
R
Lehrstuhl für Mikrobiologie der Universität München, Maria-Ward-Strasse 1a, D-80638 München, Germany.
Pajatsch
M
M
Kremmer
E
E
Böck
A
A
D001483:Base Sequence; D003001:Cloning, Molecular; D003505:Cyclodextrins / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D007700:Kinetics; D007709:Klebsiella / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D013379:Substrate Specificity; D013696:Temperature
D003505:Cyclodextrins; D006026:Glycoside Hydrolases; C048745:cyclomaltodextrinase