Identification of a novel cellulose-binding domain within the multidomain 120 kDa xylanase XynA of the hyperthermophilic bacterium Thermotoga maritima.
A segment of Thermotoga maritima strain MSB8 chromosomal DNA was isolated which encodes an endo-1,4-beta-D-xylanase, and the nucleotide sequence of the xylanase gene, designated xynA, was determined. With a half-life of about 40 min at 90 degrees C at the optimal pH of 6.2, purified recombinant XynA is one of the most thermostable xylanases known. XynA is a 1059-amino-acid (approximately 120 kDa) modular enzyme composed of an N-terminal signal peptide and five domains, in the order A1-A2-B-C1-C2. By comparison with other xylanases of family 10 of glycosyl hydrolases, the central approximately 340-amino-acid part (domain B) of XynA represents the catalytic domain. The N-terminal approximately 150-amino-acid repeated domains (A1-A2) have no significant similarity to the C-terminal approximately 170-amino-acid repeated domains (C1-C2). Cellulose-binding studies with truncated XynA derivatives and hybrid proteins indicated that the C-terminal repeated domains mediate the binding of XynA to microcrystalline cellulose and that C2 alone can also promote cellulose binding. C1 and C2 did not share amino acid sequence similarity with any other known cellulose-binding domain (CBD) and thus are CBDs of a novel type. Structurally related protein segments which are probably also CBDs were found in other multidomain xylanolytic enzymes. Deletion of the N-terminal repeated domains or of all the non-catalytic domains resulted in substantially reduced thermostability while a truncated xylanase derivative lacking the C-terminal tandem repeat was as thermostable as the full-length enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
Winterhalter
C
C
Lehrstuhl für Mikrobiologie, Technische Universität, München, Germany.
Heinrich
P
P
Candussio
A
A
Wich
G
G
Liebl
W
W
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002482:Cellulose / Q000378:metabolism*; D005798:Genes, Bacterial; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D021382:Protein Sorting Signals / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012091:Repetitive Sequences, Nucleic Acid; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D001426:Bacterial Proteins; D007527:Isoenzymes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D002482:Cellulose; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase