Role of a PA14 domain in determining substrate specificity of a glycoside hydrolase family 3 β-glucosidase from Kluyveromyces marxianus

Abstract

β-Glucosidase from Kluyveromyces marxianus (KmBglI) belongs to the GH3 (glycoside hydrolase family 3). The enzyme is particularly unusual in that a PA14 domain (pf07691), for which a carbohydrate-binding role has been claimed, is inserted into the catalytic core sequence. In the present study, we determined the enzymatic properties and crystal structure of KmBglI in complex with glucose at a 2.55 A (1 A=0.1 nm) resolution. A striking characteristic of KmBglI was that the enzyme activity is essentially limited to disaccharides, and when trisaccharides were used as the substrates the activity was drastically decreased. This chain-length specificity is in sharp contrast with the preferred action on oligosaccharides of barley β-D-glucan glucohydrolase (ExoI), which does not have a PA14 domain insertion. The structure of subsite (-1) of KmBglI is almost identical with that of Thermotoga neapolitana β-glucosidase and is also similar to that of ExoI, however, the structures of subsite (+1) significantly differ among them. In KmBglI, the loops extending from the PA14 domain cover the catalytic pocket to form subsite (+1), and hence simultaneously become a steric hindrance that could limit the chain length of the substrates to be accommodated. Mutational studies demonstrated the critical role of the loop regions in determining the substrate specificity. The active-site formation mediated by the PA14 domain of KmBglI invokes α-complementation of β-galactosidase exerted by its N-terminal domain, to which the PA14 domain shows structural resemblance. The present study is the first which reveals the structural basis of the interaction between the PA14 domain and a carbohydrate.

The structure of DesR from Streptomyces venezuelae, a β‐glucosidase involved in macrolide activation

“…Unlike DesR, the K. marxianus b-glucosidase functions on disaccharides and, to a limited extent, on trisaccharides. 20 A superposition of the DesR and the K. marxianus b-glucosidase structures is shown in Figure 4(b). Both structures were solved in the presence of D-glucose, and the carbohydrate moieties are situated in nearly identical positions in the active sites of the two enzymes.…”

“…The recent report of the K. marxianus b-glucosidase structure in 2010 highlighted the fact that the ''basic'' GH3 family member core can be decorated by two additional motifs. 20 This structural analysis was the first to demonstrate the insertion of a PA14 domain into the core unit of a GH3 glycoside hydrolase. Unlike DesR, the K. marxianus b-glucosidase functions on disaccharides and, to a limited extent, on trisaccharides.…”

“…Likewise, the structure of the K. marxianus b-glucosidase was solved only in the presence of a monosaccharide, and the PA14 domain, which was implicated in sugar binding via site-directed mutagenesis, may adopt a different orientation when a disaccharide enters the active site cleft. 20 The DesR structure described here represents the first model of a GH3 family member that functions on macrolide antibiotics. Given that, we aligned the DesR amino acid sequence against those for the homologs from the narbomycin, chalcomycin, oleandomycin, lankamycin, and erythromycin biosynthetic clusters using ClustalW2 21 and the available GenBank sequences (Supporting Information Fig.…”

Caracterização bioquímica e biofísica da enzima β-glicosidase Bgl1 de Aspergillus niger e avaliação de potenciais biomassas para produção de bioetanol

“…KmBgl1 apresenta uma inserção de um quarto domínio, PA14 (resíduos 392-559) entre o (α/) 6 -sanduíche 60 . O domínio do tipo barril-(/α) 8 , o (α/) 6 -sanduíche e o domínio FnIII são conectados, respectivamente, pelo linker 1 e linker 2.…”

“…Aspergillus niger (AnBgl1) 49 ; a -glicosidase 1 termoestável de Thermoascus aurantiacus (TaBgl1) 65 ; -glicosidase de Hypocrea jecorina (HjBgl1) 66 ; β-glicosidase de Thermotoga neapolitana (TnBgl3B) 58 , -glicosidase 1 de Kluyveromyces marxianus (KmBgl1) 60 e a exo--1,3-1,4-glucanse de Hordeum vulgare subsp. vulgare (ExoI) 59 .…”