Structure-function studies of two polysaccharide-degrading enzymes:

Bacillus stearothermophilus alfa-amylase and Trichoderma reesei cellobiohydrolase II: Dissertation

Research output: ThesisDissertationCollection of Articles

Abstract

The structure-function relationships of two polysaccharide-degrading enzymes were studied by applying two different protein engineering methods. The whole B. stearothermophilus alfa-amylase gene was subjected to random mutagenesis developed in this study. Nearly 100 diff erent alfa-amylase point mutants were produced. The enzymatic activities and location of the mutation(s) were determined and the data obtained was compared to the constructed structural model of alfa-amylase. A reasonably good overall correlation could be drawn between the mutant data and the model. Two areas of the alfa-amylase structural model were identified as being important for the activity on polymeric substrate: the open active site cleft situated between domains A and B and containing conserved amino acids known to be important for catalysis and multiple binding of glucose units in other alfa-amylases; and an interface between the catalytic domain A and domain C about 30 Å away from the active site groove. The three-dimensional structure of T. reesei cellobiohydrolase II (CBHII) catalytic domain was available. The catalytic domain has an alfa/beta barrel fold similar to a-amylase catalytic domain A. Two stable surface loops generate a 20 Å long tunnel for substrate binding and catalysis. The active site tunnel contains four defined binding sites (A-D) for glucosyl units and a putative binding site F at the entrance of the tunnel. CBHII is an inverting enzyme in which D221, situated between subsites B and C, acts as a proton donor. D175 lying next to D221 either stabilizes hypothetical carbonium ion intermediates or facilitates the protonation of D221, or both. The base has not yet been identified although the role has been attributed to D401. Site-directed mutagenesis was used to study the role of three amino acids in the active site of CBHII. Y169, located at site B close enough to interact with both D175 and the sugar hydroxyl at site B, was mutagenised to phenylalanine. The Y169F mutant showed increased binding but reduced catalytic rate on small soluble cello-oligosaccharides. These data suggest that Y169 helps to distort the glucose ring into more reactive conformation. In addition, the pH-activity profile of Y169F mutant declines at low pH, suggesting that Y169 also affects the protonation state of the active site carboxylates, D175 and D221. The tryptophan residues at subsites A and F were also mutated. In both cases removal of the indole ring affected the catalytic rate. The tight binding of an intact glucose ring in binding site A seems to be essential for efficient catalysis and is partly dictated by the W135. It was also shown that CBHII active site tunnel contains at least one additional binding site (F) at the mouth of the tunnel. It is plausible that site F is important for the breakdown of crystalline cellulose.
Original languageEnglish
QualificationDoctor Degree
Awarding Institution
  • University of Helsinki
Supervisors/Advisors
  • Lehtovaara, Päivi, Supervisor, External person
Award date2 Aug 1996
Place of PublicationEspoo
Publisher
Print ISBNs951-38-4935-X
Publication statusPublished - 1996
MoE publication typeG5 Doctoral dissertation (article)

Fingerprint

Cellulose 1,4-beta-Cellobiosidase
Geobacillus stearothermophilus
Trichoderma
alpha-Amylases
Polysaccharides
Catalytic Domain
Enzymes
Catalysis
Binding Sites
Structural Models
Glucose
Protein Engineering
Amino Acids
Amylases
Site-Directed Mutagenesis
Phenylalanine
Oligosaccharides
Cellulose
Tryptophan
Mutagenesis

Keywords

  • enzymes
  • carbohydrates
  • amylase
  • cellulase
  • protein structure
  • cellobiohydrolase
  • random mutagenesis
  • side-directed mutagenesis
  • Bacillus stearothermophilus
  • Trichoderma reesei
  • theses

Cite this

@phdthesis{557ea083076448c38c841e659e21faa1,
title = "Structure-function studies of two polysaccharide-degrading enzymes:: Bacillus stearothermophilus alfa-amylase and Trichoderma reesei cellobiohydrolase II: Dissertation",
abstract = "The structure-function relationships of two polysaccharide-degrading enzymes were studied by applying two different protein engineering methods. The whole B. stearothermophilus alfa-amylase gene was subjected to random mutagenesis developed in this study. Nearly 100 diff erent alfa-amylase point mutants were produced. The enzymatic activities and location of the mutation(s) were determined and the data obtained was compared to the constructed structural model of alfa-amylase. A reasonably good overall correlation could be drawn between the mutant data and the model. Two areas of the alfa-amylase structural model were identified as being important for the activity on polymeric substrate: the open active site cleft situated between domains A and B and containing conserved amino acids known to be important for catalysis and multiple binding of glucose units in other alfa-amylases; and an interface between the catalytic domain A and domain C about 30 {\AA} away from the active site groove. The three-dimensional structure of T. reesei cellobiohydrolase II (CBHII) catalytic domain was available. The catalytic domain has an alfa/beta barrel fold similar to a-amylase catalytic domain A. Two stable surface loops generate a 20 {\AA} long tunnel for substrate binding and catalysis. The active site tunnel contains four defined binding sites (A-D) for glucosyl units and a putative binding site F at the entrance of the tunnel. CBHII is an inverting enzyme in which D221, situated between subsites B and C, acts as a proton donor. D175 lying next to D221 either stabilizes hypothetical carbonium ion intermediates or facilitates the protonation of D221, or both. The base has not yet been identified although the role has been attributed to D401. Site-directed mutagenesis was used to study the role of three amino acids in the active site of CBHII. Y169, located at site B close enough to interact with both D175 and the sugar hydroxyl at site B, was mutagenised to phenylalanine. The Y169F mutant showed increased binding but reduced catalytic rate on small soluble cello-oligosaccharides. These data suggest that Y169 helps to distort the glucose ring into more reactive conformation. In addition, the pH-activity profile of Y169F mutant declines at low pH, suggesting that Y169 also affects the protonation state of the active site carboxylates, D175 and D221. The tryptophan residues at subsites A and F were also mutated. In both cases removal of the indole ring affected the catalytic rate. The tight binding of an intact glucose ring in binding site A seems to be essential for efficient catalysis and is partly dictated by the W135. It was also shown that CBHII active site tunnel contains at least one additional binding site (F) at the mouth of the tunnel. It is plausible that site F is important for the breakdown of crystalline cellulose.",
keywords = "enzymes, carbohydrates, amylase, cellulase, protein structure, cellobiohydrolase, random mutagenesis, side-directed mutagenesis, Bacillus stearothermophilus, Trichoderma reesei, theses",
author = "Anu Koivula",
note = "Project code: B6SU00010",
year = "1996",
language = "English",
isbn = "951-38-4935-X",
series = "VTT Publications",
publisher = "VTT Technical Research Centre of Finland",
number = "277",
address = "Finland",
school = "University of Helsinki",

}

Structure-function studies of two polysaccharide-degrading enzymes: Bacillus stearothermophilus alfa-amylase and Trichoderma reesei cellobiohydrolase II: Dissertation. / Koivula, Anu.

Espoo : VTT Technical Research Centre of Finland, 1996. 97 p.

Research output: ThesisDissertationCollection of Articles

TY - THES

T1 - Structure-function studies of two polysaccharide-degrading enzymes:

T2 - Bacillus stearothermophilus alfa-amylase and Trichoderma reesei cellobiohydrolase II: Dissertation

AU - Koivula, Anu

N1 - Project code: B6SU00010

PY - 1996

Y1 - 1996

N2 - The structure-function relationships of two polysaccharide-degrading enzymes were studied by applying two different protein engineering methods. The whole B. stearothermophilus alfa-amylase gene was subjected to random mutagenesis developed in this study. Nearly 100 diff erent alfa-amylase point mutants were produced. The enzymatic activities and location of the mutation(s) were determined and the data obtained was compared to the constructed structural model of alfa-amylase. A reasonably good overall correlation could be drawn between the mutant data and the model. Two areas of the alfa-amylase structural model were identified as being important for the activity on polymeric substrate: the open active site cleft situated between domains A and B and containing conserved amino acids known to be important for catalysis and multiple binding of glucose units in other alfa-amylases; and an interface between the catalytic domain A and domain C about 30 Å away from the active site groove. The three-dimensional structure of T. reesei cellobiohydrolase II (CBHII) catalytic domain was available. The catalytic domain has an alfa/beta barrel fold similar to a-amylase catalytic domain A. Two stable surface loops generate a 20 Å long tunnel for substrate binding and catalysis. The active site tunnel contains four defined binding sites (A-D) for glucosyl units and a putative binding site F at the entrance of the tunnel. CBHII is an inverting enzyme in which D221, situated between subsites B and C, acts as a proton donor. D175 lying next to D221 either stabilizes hypothetical carbonium ion intermediates or facilitates the protonation of D221, or both. The base has not yet been identified although the role has been attributed to D401. Site-directed mutagenesis was used to study the role of three amino acids in the active site of CBHII. Y169, located at site B close enough to interact with both D175 and the sugar hydroxyl at site B, was mutagenised to phenylalanine. The Y169F mutant showed increased binding but reduced catalytic rate on small soluble cello-oligosaccharides. These data suggest that Y169 helps to distort the glucose ring into more reactive conformation. In addition, the pH-activity profile of Y169F mutant declines at low pH, suggesting that Y169 also affects the protonation state of the active site carboxylates, D175 and D221. The tryptophan residues at subsites A and F were also mutated. In both cases removal of the indole ring affected the catalytic rate. The tight binding of an intact glucose ring in binding site A seems to be essential for efficient catalysis and is partly dictated by the W135. It was also shown that CBHII active site tunnel contains at least one additional binding site (F) at the mouth of the tunnel. It is plausible that site F is important for the breakdown of crystalline cellulose.

AB - The structure-function relationships of two polysaccharide-degrading enzymes were studied by applying two different protein engineering methods. The whole B. stearothermophilus alfa-amylase gene was subjected to random mutagenesis developed in this study. Nearly 100 diff erent alfa-amylase point mutants were produced. The enzymatic activities and location of the mutation(s) were determined and the data obtained was compared to the constructed structural model of alfa-amylase. A reasonably good overall correlation could be drawn between the mutant data and the model. Two areas of the alfa-amylase structural model were identified as being important for the activity on polymeric substrate: the open active site cleft situated between domains A and B and containing conserved amino acids known to be important for catalysis and multiple binding of glucose units in other alfa-amylases; and an interface between the catalytic domain A and domain C about 30 Å away from the active site groove. The three-dimensional structure of T. reesei cellobiohydrolase II (CBHII) catalytic domain was available. The catalytic domain has an alfa/beta barrel fold similar to a-amylase catalytic domain A. Two stable surface loops generate a 20 Å long tunnel for substrate binding and catalysis. The active site tunnel contains four defined binding sites (A-D) for glucosyl units and a putative binding site F at the entrance of the tunnel. CBHII is an inverting enzyme in which D221, situated between subsites B and C, acts as a proton donor. D175 lying next to D221 either stabilizes hypothetical carbonium ion intermediates or facilitates the protonation of D221, or both. The base has not yet been identified although the role has been attributed to D401. Site-directed mutagenesis was used to study the role of three amino acids in the active site of CBHII. Y169, located at site B close enough to interact with both D175 and the sugar hydroxyl at site B, was mutagenised to phenylalanine. The Y169F mutant showed increased binding but reduced catalytic rate on small soluble cello-oligosaccharides. These data suggest that Y169 helps to distort the glucose ring into more reactive conformation. In addition, the pH-activity profile of Y169F mutant declines at low pH, suggesting that Y169 also affects the protonation state of the active site carboxylates, D175 and D221. The tryptophan residues at subsites A and F were also mutated. In both cases removal of the indole ring affected the catalytic rate. The tight binding of an intact glucose ring in binding site A seems to be essential for efficient catalysis and is partly dictated by the W135. It was also shown that CBHII active site tunnel contains at least one additional binding site (F) at the mouth of the tunnel. It is plausible that site F is important for the breakdown of crystalline cellulose.

KW - enzymes

KW - carbohydrates

KW - amylase

KW - cellulase

KW - protein structure

KW - cellobiohydrolase

KW - random mutagenesis

KW - side-directed mutagenesis

KW - Bacillus stearothermophilus

KW - Trichoderma reesei

KW - theses

M3 - Dissertation

SN - 951-38-4935-X

T3 - VTT Publications

PB - VTT Technical Research Centre of Finland

CY - Espoo

ER -