The investigation of type-specific features of the copper coordinating AA9 proteins and their effect on the interaction with crystalline cellulose using molecular dynamics studies
- Authors: Moses, Vuyani
- Date: 2018
- Subjects: Copper proteins , Cellulose , Molecular dynamics , Cellulose -- Biodegradation , Bioinformatics
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/58327 , vital:27230
- Description: AA9 proteins are metallo-enzymes which are crucial for the early stages of cellulose degradation. AA9 proteins have been suggested to cleave glycosidic bonds linking cellulose through the use of their Cu2+ coordinating active site. AA9 proteins possess different regioselectivities depending on the resulting cleavage they form and as result, are grouped accordingly. Type 1 AA9 proteins cleave the C1 carbon of cellulose while Type 2 AA9 proteins cleave the C4 carbon and Type 3 AA9 proteins cleave either C1 or C4 carbons. The steric congestion of the AA9 active site has been proposed to be a contributor to the observed regioselectivity. As such, a bioinformatics characterisation of type-specific sequence and structural features was performed. Initially AA9 protein sequences were obtained from the Pfam database and multiple sequence alignment was performed. The sequences were phylogenetically characterised and sequences were grouped into their respective types and sub-groups were identified. A selection analysis was performed on AA9 LPMO types to determine the selective pressure acting on AA9 protein residues. Motif discovery was then performed to identify conserved sequence motifs in AA9 proteins. Once type-specific sequence features were identified structural mapping was performed to assess possible effects on substrate interaction. Physicochemical property analysis was also performed to assess biochemical differences between AA9 LPMO types. Molecular dynamics (MD) simulations were then employed to dynamically assess the consequences of the discovered type-specific features on AA9-cellulose interaction. Due to the absence of AA9 specific force field parameters MD simulations were not readily applicable. As a result, Potential Energy Surface (PES) scans were performed to evaluate the force field parameters for the AA9 active site using the PM6 semi empirical approach and least squares fitting. A Type 1 AA9 active site was constructed from the crystal structure 4B5Q, encompassing only the Cu2+ coordinating residues, the Cu2+ ion and two water residues. Due to the similarity in AA9 active sites, the Type force field parameters were validated on all three AA9 LPMO types. Two MD simulations for each AA9 LPMO types were conducted using two separate Lennard-Jones parameter sets. Once completed, the MD trajectories were analysed for various features including the RMSD, RMSF, radius of gyration, coordination during simulation, hydrogen bonding, secondary structure conservation and overall protein movement. Force field parameters were successfully evaluated and validated for AA9 proteins. MD simulations of AA9 proteins were able to reveal the presence of unique type-specific binding modes of AA9 active sites to cellulose. These binding modes were characterised by the presence of unique type-specific loops which were present in Type 2 and 3 AA9 proteins but not in Type 1 AA9 proteins. The loops were found to result in steric congestion that affects how the Cu2+ ion interacts with cellulose. As a result, Cu2+ binding to cellulose was observed for Type 1 and not Type 2 and 3 AA9 proteins. In this study force field parameters have been evaluated for the Type 1 active site of AA9 proteins and this parameters were evaluated on all three types and binding. Future work will focus on identifying the nature of the reactive oxygen species and performing QM/MM calculations to elucidate the reactive mechanism of all three AA9 LPMO types.
- Full Text:
- Date Issued: 2018
- Authors: Moses, Vuyani
- Date: 2018
- Subjects: Copper proteins , Cellulose , Molecular dynamics , Cellulose -- Biodegradation , Bioinformatics
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/58327 , vital:27230
- Description: AA9 proteins are metallo-enzymes which are crucial for the early stages of cellulose degradation. AA9 proteins have been suggested to cleave glycosidic bonds linking cellulose through the use of their Cu2+ coordinating active site. AA9 proteins possess different regioselectivities depending on the resulting cleavage they form and as result, are grouped accordingly. Type 1 AA9 proteins cleave the C1 carbon of cellulose while Type 2 AA9 proteins cleave the C4 carbon and Type 3 AA9 proteins cleave either C1 or C4 carbons. The steric congestion of the AA9 active site has been proposed to be a contributor to the observed regioselectivity. As such, a bioinformatics characterisation of type-specific sequence and structural features was performed. Initially AA9 protein sequences were obtained from the Pfam database and multiple sequence alignment was performed. The sequences were phylogenetically characterised and sequences were grouped into their respective types and sub-groups were identified. A selection analysis was performed on AA9 LPMO types to determine the selective pressure acting on AA9 protein residues. Motif discovery was then performed to identify conserved sequence motifs in AA9 proteins. Once type-specific sequence features were identified structural mapping was performed to assess possible effects on substrate interaction. Physicochemical property analysis was also performed to assess biochemical differences between AA9 LPMO types. Molecular dynamics (MD) simulations were then employed to dynamically assess the consequences of the discovered type-specific features on AA9-cellulose interaction. Due to the absence of AA9 specific force field parameters MD simulations were not readily applicable. As a result, Potential Energy Surface (PES) scans were performed to evaluate the force field parameters for the AA9 active site using the PM6 semi empirical approach and least squares fitting. A Type 1 AA9 active site was constructed from the crystal structure 4B5Q, encompassing only the Cu2+ coordinating residues, the Cu2+ ion and two water residues. Due to the similarity in AA9 active sites, the Type force field parameters were validated on all three AA9 LPMO types. Two MD simulations for each AA9 LPMO types were conducted using two separate Lennard-Jones parameter sets. Once completed, the MD trajectories were analysed for various features including the RMSD, RMSF, radius of gyration, coordination during simulation, hydrogen bonding, secondary structure conservation and overall protein movement. Force field parameters were successfully evaluated and validated for AA9 proteins. MD simulations of AA9 proteins were able to reveal the presence of unique type-specific binding modes of AA9 active sites to cellulose. These binding modes were characterised by the presence of unique type-specific loops which were present in Type 2 and 3 AA9 proteins but not in Type 1 AA9 proteins. The loops were found to result in steric congestion that affects how the Cu2+ ion interacts with cellulose. As a result, Cu2+ binding to cellulose was observed for Type 1 and not Type 2 and 3 AA9 proteins. In this study force field parameters have been evaluated for the Type 1 active site of AA9 proteins and this parameters were evaluated on all three types and binding. Future work will focus on identifying the nature of the reactive oxygen species and performing QM/MM calculations to elucidate the reactive mechanism of all three AA9 LPMO types.
- Full Text:
- Date Issued: 2018
The large scale bioinformatics analysis of auxiliary activity family 9 enzymes
- Authors: Moses, Vuyani
- Date: 2014
- Subjects: Bioinformatics -- Analysis , Cellulose -- Biodegradation , Biomass energy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4145 , http://hdl.handle.net/10962/d1016356
- Description: Biofuels have been proposed to be a suitable replacement to the already depleting fossil fuels. The complex structures of plant biomasses present a challenge the production of biofuels due to recalcitrance. The complex cellulose structure and hydrogen bonding between repeat units of cellulose is believed to be a major contributor to the recalcitrance of cellulose. Fungal organisms come equipped with various oxidative enzymes involved in degradation of plant biomass. The exact mechanism of cellulose degradation remains elusive. The GH61 is a group of proteins which are PMOs. GH61 sequences where previously described as endoglucanases due to weak endoglucanase activity. These enzymes were later found not possess any enzyme activity of their own however they could enhance the activity of other cellulose degrading enzymes. As a result reclassification of these enzymes as AA9 has been implemented. AA9 proteins have been reported to share structural homology with the bacterial AA10 group of enzymes. Based on cleavage products that are produced when AA9 proteins interact with cellulose, AA9 proteins have been grouped into three types. To date the exact mechanism and the sequence and structural basis for differentiating between the various AA9 types remains unknown. Using various bionformatic techniques sequence and structural elements were identified for distinguishing between the AA9 types. A large dataset of sequences was obtained from the Pfam database from UNIPROT entries. Due to high divergence of AA9 sequences, a smaller dataset with the more divergent sequences removed was created. The inclusion of the reference sequences to the data set was done to observe which sequences belong to a certain type. Phylogenetic analysis was able to group AA9 proteins into three distinct groups. MSA and motif analysis revealed that the N-Terminus of these proteins is mostly responsible for type specificity. Structural analysis of AA9 PDB structures and homology models allowed the effect of physicochemical properties to be gauged structurally. The presence of 310 helices and aromatic residues the surface of AA9 sequences is an observation which still warrants further investigation.
- Full Text:
- Date Issued: 2014
- Authors: Moses, Vuyani
- Date: 2014
- Subjects: Bioinformatics -- Analysis , Cellulose -- Biodegradation , Biomass energy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4145 , http://hdl.handle.net/10962/d1016356
- Description: Biofuels have been proposed to be a suitable replacement to the already depleting fossil fuels. The complex structures of plant biomasses present a challenge the production of biofuels due to recalcitrance. The complex cellulose structure and hydrogen bonding between repeat units of cellulose is believed to be a major contributor to the recalcitrance of cellulose. Fungal organisms come equipped with various oxidative enzymes involved in degradation of plant biomass. The exact mechanism of cellulose degradation remains elusive. The GH61 is a group of proteins which are PMOs. GH61 sequences where previously described as endoglucanases due to weak endoglucanase activity. These enzymes were later found not possess any enzyme activity of their own however they could enhance the activity of other cellulose degrading enzymes. As a result reclassification of these enzymes as AA9 has been implemented. AA9 proteins have been reported to share structural homology with the bacterial AA10 group of enzymes. Based on cleavage products that are produced when AA9 proteins interact with cellulose, AA9 proteins have been grouped into three types. To date the exact mechanism and the sequence and structural basis for differentiating between the various AA9 types remains unknown. Using various bionformatic techniques sequence and structural elements were identified for distinguishing between the AA9 types. A large dataset of sequences was obtained from the Pfam database from UNIPROT entries. Due to high divergence of AA9 sequences, a smaller dataset with the more divergent sequences removed was created. The inclusion of the reference sequences to the data set was done to observe which sequences belong to a certain type. Phylogenetic analysis was able to group AA9 proteins into three distinct groups. MSA and motif analysis revealed that the N-Terminus of these proteins is mostly responsible for type specificity. Structural analysis of AA9 PDB structures and homology models allowed the effect of physicochemical properties to be gauged structurally. The presence of 310 helices and aromatic residues the surface of AA9 sequences is an observation which still warrants further investigation.
- Full Text:
- Date Issued: 2014
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