Domain architecture divergence leads to functional divergence in binding and catalytic domains of bacterial and fungal cellobiohydrolases

Abstract

Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD. Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD. Cellobiohydrolases (CBHs) play key roles in degradation of crystalline cellulose, which is the homopolymer of β-1,4-linked glucose and fundamental component of plant cell wall (1Teeri T.T. Crystalline cellulose degradation: new insight into the function of cellobiohydrolases.Trends Biotechnol. 1997; 15: 160-16710.1016/S0167-7799(97)01032-9Abstract Full Text PDF Scopus (534) Google Scholar). High hydrolytic activity of CBHs against crystalline cellulose is achieved by the unique structure of the catalytic domain (CD), which consists of tunnel-shaped substrate-binding sites covered by loops (2Davies G. Henrissat B. Structures and mechanisms of glycosyl hydrolases.Structure. 1995; 3 (8535779): 853-85910.1016/S0969-2126(01)00220-9Abstract Full Text Full Text PDF PubMed Scopus (1610) Google Scholar). In addition, many CBHs also have the cellulose-binding domain (CBD), and the CD and CBD are connected by the linker region (or domain). The CD and CBD are classified into glycoside hydrolase (GH) and carbohydrate binding module (CBM) families, respectively, according to the amino acid sequences (3Lombard V. Golaconda Ramulu H. Drula E. Coutinho P.M. Henrissat B. The carbohydrate-active enzymes database (CAZy) in 2013.Nucleic Acids Res. 2014; 42 (24270786): D490-D49510.1093/nar/gkt1178Crossref PubMed Scopus (4138) Google Scholar). Although cellulases are classified into GH families 5–12, 44, 45, 48, 51, 74, 124, and 148, CBHs are only included in the members of GH6, 7, 9, and 48. The CBMs, which have flat surfaces for cellulose binding (called as type A CBM), are divided into CBM 1, 2, 3, 5, and 10 (4Boraston A.B. Bolam D.N. Gilbert H.J. Davies G.J. Carbohydrate-binding modules: fine-tuning polysaccharide recognition.Biochem. J. 2004; 382 (15214846): 769-78110.1042/BJ20040892Crossref PubMed Scopus (1509) Google Scholar). Cellulose is the most abundant biomass on earth and an important carbon source for fungi and bacteria. Cellulose degradation system of fungi has been well-known, and they produce many kinds of multidomain cellulases. Synergistic hydrolytic reactions between GH7 and GH6 CBHs (5Henrissat B. Driguez H. Viet C. Schülein of cellulases from Trichoderma reesei in the degradation of Scopus Google or CBHs and have been in important cellulose degradation system of is which is the of carbohydrate enzymes to the cell The system is by and only 9, and cellulases are as and PubMed Scopus Google Scholar). degradation system of is similar to the an Cellulomonas fimi GH6 and CBHs C. E. Cellulomonas fimi in in for the Biotechnol. PubMed Scopus Google Scholar). In the of crystalline cellulose cellulases on the cellulose However, the not cellulases is of the of the cellulose in the a a cellulose into the catalytic it In and from the cellulose surface The unique function of is a on cellulose surface with processive of the cellulose into the The of has been directly observed by single-molecule imaging the GH7 which is a unique for the was by hydrolytic of on cellulose PubMed Scopus Google High processive of Trichoderma reesei cellobiohydrolase on crystalline Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, the between of the structure of the CD and has been and hydrolase is directly to binding PubMed Scopus Google H. between and hydrolytic of cellobiohydrolases at the surface of crystalline 2014; PubMed Scopus Google Scholar). Although GH6 CBHs are enzymes in fungi and the of GH6 from only an Trichoderma reesei (TrCel6A) has been observed by single-molecule fluorescence imaging H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google Scholar). GH7 cellulose from and GH6 from of the cellulose they show in the processive GH6 CBHs from fungi and are classified into the but are different in many the structure of the substrate-binding of bacterial CD is longer that of fungi CD C. J. H. The structure of a bacterial the catalytic of the GH6 cellobiohydrolase PubMed Scopus Google Scholar). bacterial GH6 was to processive and GH6 the CBD and linker region are TrCel6A has connected to CD by a glycosylated linker region The linker region is to and the of on the linker with the cellulose surface is by the J. in enzymes to PubMed Scopus Google Scholar). In GH6 from the bacterium C. fimi (CfCel6B) has and three fibronectin type 3 domains as a linker between CD and have flat surface with and are to on the surface of the crystalline cellulose J. H. Henrissat B. of the of a bacterial and cellulose-binding domain to crystalline J. PubMed Scopus Google J. J. T.T. The binding and of and 3 cellulose binding PubMed Scopus Google Scholar). CfCel6B is previously and found as the that is similar to the II from reesei of E. Cellobiohydrolase A from the bacterium Cellulomonas fimi is a to Trichoderma reesei PubMed Scopus Google Scholar). However, the of the different domain on the of cellulose are In by using single-molecule fluorescence we observed binding and dissociation of CfCel6B and domains and on the crystalline cellulose to the of CBD and Furthermore, on the crystalline cellulose Combined with crystal of the CD from bacterial and GH6 CBHs and we between of the structure of the CD and processivity. Furthermore, in phylogenetic tree analysis, the from and fungi The CBHs have glycosylated linker and and bacterial CBHs have for the domain the of domain between bacterial and of bacterial CBHs for initial binding on cellulose by glycosylated of results the of a between bacterial CBHs and In to single-molecule fluorescence on the surface of CfCel6B and domains. We as (CD), and In addition, a of of which catalytic acid was to was as a of processive with of in and using cellulose or acid the of the with hydrolytic of and compared with crystalline cellulose at the rate of and showed hydrolytic rate the hydrolytic rate of was very that not have cellulose hydrolytic activity. we compared hydrolytic for and at of crystalline cellulose to the and by the with The for and and and for and and for and that hydrolytic activity was also on and enzymes for results that and with not the hydrolytic activity and of CfCel6B against crystalline cellulose. the binding rate constant for CD, and enzymes of on the with crystalline cellulose to fluorescence from The of enzymes to the cellulose and to the surface results that of the enzymes have that the surface of crystalline cellulose. The of as of divided by of cellulose and We not directly the of in the cellulose from the fluorescence with of of the of of we the of of for showed at of for CD, and CBD showed which to of in cellulose The and The for and CBD and and of that for the of for CD showed a at which was of that for results that of CfCel6B is on the binding of the and and CD not However, CD and CBD connected by domains to the for is the of for CD and Synergistic binding between CBD with linker region and CD has been also observed in TrCel6A H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google Scholar). we of binding on cellulose of binding by of in and CBD and as previously for TrCel6A H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google Scholar). results that at different binding of the CD, the for the of the was to the of and slow of dissociation rate constant and for and The of fast and slow and for CD and and compared with for In and of fast for and CBD and In addition, of slow for and CBD and between and CBD but three compared with for results that of CfCel6B is by CD. We also found that CD showed much higher of fast dissociation that of slow dissociation was unique for CD the CD, and the of slow dissociation for the three The of the binding rate constant to the fast and slow and from the in 3 and the of fast and slow by the in the of the dissociation constant for the fast and slow and from the of to for showed the was that of for The of or for and CBD and higher of the for CD was higher that of of the of The of for CD and was results that CBD to the of fast and slow of of and dissociation or and are the fast and slow of the dissociation rate by the of the of binding with a or in 3 divided into and by using the of fast and slow of or and as and of fast and slow from the of the of The and are the fast and slow of the dissociation rate by the of the of binding with a The of fast and slow from the of the of The in 3 divided into and by using the of fast and slow of The and as and in a new rate was from the between and of and We higher and rate for binding and dissociation to achieve higher and for the and of for and CD with of and for and and for CD and the of for and with a and the and 10 and of showed the 5, and the in 10 5, results that that showed a to and that showed a are a of and on the cellulose the of for was to for the or the longer 10 The constant of for the was and that for the was the for TrCel6A in previously H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google of of for was by and are with the and using only the 10 The was from The of We a crystal structure of CfCel6B CD with to the between CfCel6B and TrCel6A The structure of CfCel6B CD was by on a GH6 from a bacterium and was as a of C. J. H. The structure of a bacterial the catalytic of the GH6 cellobiohydrolase PubMed Scopus Google Scholar). In the crystal structure of CfCel6B CD, we that the which is to the of CfCel6B and to in the single-molecule fluorescence was in the of the catalytic In the crystal CfCel6B has substrate-binding by at the as 7, is by loops 7, in which are not found in TrCel6A 7, the a of exit loops has been also found in CfCel6B and The exit in was the of the substrate-binding exit of CfCel6B is amino that of that the tunnel of CfCel6B is that of In addition, the exit in of CfCel6B showed compared with that of The of the of CfCel6B was compared with of TrCel6A and The and in CfCel6B, and which is to important to a the and catalytic on the J. of a catalytic Full Text Full Text PDF PubMed Scopus Google Scholar). The and the of CfCel6B, of the In the with the and the loops to of the in the of TrCel6A and The of the by the with has been previously for the GH6 from a structure of a cellobiohydrolase from the PubMed Scopus Google Scholar). the between the structure and function of CD, we compared of GH6 enzymes in database the enzymes with only or in the the of crystal and of CD, In the phylogenetic tree bacterial and cellulases and CBHs from and fungi different but of fungi showed domain the phylogenetic tree was on the sequences of only CD not have and many of CBHs by CD and with or glycosylated linker cellulases a glycosylated linker and that show that of J. Bolam D.N. Gilbert H.J. The type II and cellulose-binding domains of A catalytic activity against by a J. PubMed Scopus Google Scholar). the bacterial three of domain only CD, and the CD with in the or Furthermore, bacterial CBHs showed different was the and also In addition, of an CD domain classified into or the domain of CBHs from fungi and the with the glycosylated linker is the domains for The is the domain for bacterial but the is is the of domains. The CBHs on the of CD. Although bacterial CBHs not show clear of GH6 CBHs from is to a In we found that CfCel6B similar to TrCel6A and for CfCel6B and CfCel6B has different domain from that of TrCel6A H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google Scholar). the the for CfCel6B was much that for TrCel6A that the of CfCel6B to the crystalline cellulose is higher that of TrCel6A. the in the mechanisms of crystalline cellulose by we compare the kinetic parameters of as binding and and dissociation in the and H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google Scholar). single-molecule fluorescence imaging of the and of processive cellulases to which the to the crystalline cellulose 3, CfCel6B and we found fast and slow that to the on and crystal surfaces of the cellulose, H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google Scholar). that cellulose on high-affinity crystal the slow component is to the only a binding to the The of for CfCel6B and TrCel6A and 3 and and H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google The of for CfCel6B and TrCel6A and and H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google Scholar). the of or between CfCel6B and TrCel6A are However, the of slow dissociation component for CfCel6B was and much higher that for TrCel6A The higher of the slow dissociation component that CfCel6B to the surface TrCel6A. is of the that CfCel6B showed a TrCel6A. However, is not of the for binding was of the rate and similar to the for single-molecule fluorescence imaging of A from H. imaging the of a of A from Full Text Full Text PDF PubMed Scopus Google Scholar). role of the CD in the binding and dissociation of CfCel6B and TrCel6A on the cellulose we also and for CfCel6B CD 3, and crystal structure structure of CfCel6B CD was similar to that of the TrCel6A CD for the and Although in is to the the of and for CfCel6B CD similar to for TrCel6A CD in H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, the of for CfCel6B CD was not different from that for TrCel6A the binding and dissociation are not by the and loops of CfCel6B CD. The most domain for binding and dissociation of CfCel6B is CBD 3, In the the CBD showed 10 higher CD, and the was of that for results that CBD has a role in the initial of CfCel6B with crystalline cellulose. the of or between CD and CBD showed that binding of CD to or surface of crystalline cellulose is of CBD and However, the of slow dissociation component for CD was and much that for CBD which was to the for of binding for is a of the between CD and but the of binding to the surface from the binding by The CBD of CfCel6B is a of type A which is to cellulose and has a flat surface with E. structure of a cellulose-binding domain from Cellulomonas fimi by 1995; PubMed Scopus Google Scholar). of CBD in is to by an of E. 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H. and binding of A by single-molecule PubMed Google Scholar). the only of the is to at is by the of the on the surface of crystalline cellulose. and fast in the on crystalline cellulose have been also observed in and for slow and fast and H. imaging of processive movement, and dissociation of cellobiohydrolase Trichoderma reesei and domains on crystalline Full Text Full Text PDF PubMed Scopus Google Scholar). The fast to surface diffusion of cellulose as previously on the using fluorescence the diffusion of cellulases and binding domains on 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). Furthermore, an was found between of CfCel6B and TrCel6A. 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The tree was by according to and results of The structure in has been into the with the are the We members at the for with with cellobiohydrolase cellulose-binding domain catalytic domain module glycoside hydrolase glycoside hydrolase cellobiohydrolase from C. fimi glycoside hydrolase cellobiohydrolase from reesei glycoside hydrolase cellobiohydrolase from fibronectin type 3 domain of

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