Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima.
The ability of the hyperthermophilic bacterium Thermotoga maritima to grow on pectin as a sole carbon source coincides with the secretion of a pectate lyase A (PelA) in the extracellular medium. The pel A gene of T. maritima was functionally expressed in Escherichia coli as the first heterologously produced thermophilic pectinase, and purified to homogeneity. Gel filtration indicated that the native form of PelA is tetrameric. Highest activity (422 units/mg, with a K(m) of 0.06 mM) was demonstrated on polygalacturonic acid (PGA), whereas pectins with an increasing degree of methylation were degraded at a decreasing rate. In the tradition of pectate lyases, PelA demonstrated full dependency on Ca(2+) for stability and activity. The enzyme is highly thermoactive and thermostable, operating optimally at 90 degrees C and pH 9.0, with a half-life for thermal inactivation of almost 2 h at 95 degrees C, and an apparent melting temperature of 102.5 degrees C. Detailed characterization of the product formation with PGA indicated that PelA has a unique eliminative exo-cleavage pattern liberating unsaturated trigalacturonate as the major product, in contrast with unsaturated digalacturonate for other exopectate lyases known. The unique exo-acting mode of action was supported by progression profiles of PelA on oligogalacturonides (degree of polymerization, 3-8) and the examination of the bond cleavage frequencies.
Kluskens
Leon D
LD
Laboratory of Microbiology, Wageningen University, Hesselink van Suchtelenweg 4, NL-6703, CT, Wageningen, The Netherlands. leon.klukens@algemeen.wag-ur.nl
van Alebeek
Gert-Jan W M
GJ
Voragen
Alphons G J
AG
de Vos
Willem M
WM
van der Oost
John
J
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003001:Cloning, Molecular; D006358:Hot Temperature; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010368:Pectins / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D020124:Thermotoga maritima / Q000201:enzymology* / Q000254:growth & development
D001426:Bacterial Proteins; D009844:Oligosaccharides; C412763:oligogalacturonic acid; D010368:Pectins; D011133:Polysaccharide-Lyases; C023911:pectate lyase