Site-directed mutation of noncatalytic residues of Thermobifida fusca exocellulase Cel6B.
Fifteen mutant genes in six loop residues and eight mutant genes in five conserved noncatalytic active site residues of Thermobifida fusca Cel6B were constructed, cloned and expressed in Escherichia coli or Streptomyces lividans. The mutant enzymes were assayed for catalytic activity on carboxymethyl cellulose (CMC), swollen cellulose (SC), filter paper (FP), and bacterial microcrystalline cellulose (BMCC) as well as cellotetraose, cellopentaose, and 2, 4-dinitrophenyl-beta-D-cellobioside. They were also assayed for ligand binding, enzyme processivity, thermostability, and cellobiose feedback inhibition. Two double Cys mutations that formed disulfide bonds across two tunnel forming loops were found to significantly weaken binding to ligands, lower all activities, and processivity, demonstrating that the movement of these loops is important but not essential for Cel6B function. Two single mutant enzymes, G234S and G284P, had higher activity on SC and FP, and the double mutant enzyme had threefold and twofold higher activity on these substrates, respectively. However, synergism with endocellulase T. fusca Cel5A was not increased with these mutant enzymes. All mutant enzymes with lower activity on filter paper, BMCC, and SC had lower processivity. This trend was not true for CMC, suggesting that processivity in Cel6B is a key factor in the hydrolysis of insoluble and crystalline cellulose. Three mutations (E495D, H326A and W329C) located near putative glycosyl substrate subsites -2, +1 and +2, were found to significantly increase resistance to cellobiose feedback inhibition. Both the A229V and L230C mutations specifically decreased activity on BMCC, suggesting that BMCC hydrolysis has a different rate limiting step than the other substrates. Most of the mutant enzymes had reduced thermostability although Cel6B G234S maintained wild-type thermostability. The properties of the different mutant enzymes provide insight into the catalytic mechanism of Cel6B.
Zhang
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Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Irwin
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DC
Wilson
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D000192:Actinomycetales / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D002475:Cellobiose / Q000378:metabolism; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D005960:Glucosides / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009844:Oligosaccharides / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013780:Tetroses / Q000378:metabolism
C410598:2,4-dinitrophenyl-cellobioside; D001426:Bacterial Proteins; D005960:Glucosides; D009844:Oligosaccharides; D011134:Polysaccharides; D011993:Recombinant Fusion Proteins; D013780:Tetroses; D002475:Cellobiose; C019193:maltopentaose; C048468:cellotetraose; D002482:Cellulose; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase