Characterization and mode of action of an exopolygalacturonase from the hyperthermophilic bacterium Thermotoga maritima.
An intracellular pectinolytic enzyme, PelB (TM0437), from the hyperthermophilic bacterium Thermotoga maritima was functionally produced in Escherichia coli and purified to homogeneity. PelB belongs to family 28 of the glycoside hydrolases, consisting of pectin-hydrolysing enzymes. As one of the few bacterial exopolygalacturonases, it is able to remove monogalacturonate units from the nonreducing end of polygalacturonate. Detailed characterization of the enzyme showed that PelB is highly thermo-active and thermostable, with a melting temperature of 105 degrees C and a temperature optimum of 80 degrees C, the highest described to date for hydrolytic pectinases. PelB showed increasing activity on oligosaccharides with an increasing degree of polymerization. The highest activity was found on the pentamer (1000 U.mg(-1)). In addition, the affinity increased in conjunction with the length of the oligoGalpA chain. PelB displayed specificity for saturated oligoGalpA and was unable to degrade unsaturated or methyl-esterified oligoGalpA. Analogous to the exopolygalacturonase from Aspergillus tubingensis, it showed low activity with xylogalacturonan. Calculations on the subsite affinity revealed the presence of four subsites and a high affinity for GalpA at subsite +1, which is typical of exo-active enzymes. The physiological role of PelB and the previously characterized exopectate lyase PelA is discussed.
Kluskens
Leon D
LD
Laboratory of Microbiology, Wageningen University, the Netherlands.
van Alebeek
Gert-Jan W M
GJ
Walther
Jasper
J
Voragen
Alphons G J
AG
de Vos
Willem M
WM
van der Oost
John
J
D000595:Amino Acid Sequence; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D011133:Polysaccharide-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D006026:Glycoside Hydrolases; C020136:exopolygalacturonase; D011133:Polysaccharide-Lyases; C023911:pectate lyase