Contribution of (β/α)4 subdomains to the stability and activity of β-glycosidases GH1 with (β/α)8 barrel structure

Author(s)

Maira Artischeff Frutuoso

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2019-10-04

Examining board members

Sandro Roberto Marana; Cristiane Rodrigues Guzzo Carvalho; Roberto Kopke Salinas; Ivarne Luis dos Santos Tersariol

• Advisor: Sandro Roberto Marana

Abstract

Domains are stable and independent folding units that compose the protein structure (Richardson, 1981; Dootlitle, 1995; Porter e Rose, 2012). Interestingly, the application of this definition in the analysis of (β/α)8 barrel proteins reveals two domains corresponding to the N- and C-terminal halves (Porter and Rose, 2012), a perspective that contrast with the common assumption that those are single domain proteins. Based on that, assuming that the N- and C-terminal halves of the (β/α)8 barrel proteins are stable and independent, the objective of this project was to understand how the individual properties of the (β/α)4 subdomains combine and define the stability and catalytic activity of the β-glycosidases GH1, which exhibit (β/α)8 structure. The β-glycosidase bglThm from Thermotoga maritima (thermophile bacteria), bglA and bglB from Paenibacillus polymyxa (mesophile bacteria) and six chimeric proteins combining the (β/ α)4 subdomains from two of those enzymes (NbglThm-CbglB, NbglB-CbglThm, NbglThm-CbglA, NbglA-CbglThm, NbglA-CbglB e NbglB-CbglA) were used as experimental models. bglA, bglB and bglThm were produced as recombinant proteins and successfully purified. The chimera NbglA-CbglThm was the only one that was viably produced and purified. Tryptophan fluorescence and circular dichroism spectra indicated that the wild-type and the chimeric β-glycosidases were folded. NbglA-CbglThm has a secondary structure composition similar to bglA. On the other hand, access of the tryptophan residues of NbglA-CbglThm to the quencher acrylamide is comparable to bglThm. The melting temperature (Tm) and the thermal inactivation rate constant (at 47°C) of the NbglA-CbglThm are intermediary to bglA and bglThm. Hence, the chimera thermostability is a balance of the parental enzymes stabilities. The catalytic activity of NbglA-CbglThm does not depend on the pH, whereas bglA and bglThm exhibited a typical bell shaped curve. In addition, NbglA-CbglThm activity is lower than the parental enzymes, as showed the kcat for three different substrates. Considering that the active site properties of the β-glycosidases rely on the relative positioning of at least ten residues, such changes are not surprising. The Km of NbglA-CbglThm for substrates pNPβ-Gluco and pNPβ-Fuco are intermediary to bglA and bglThm. Finally, the substrate specificity of the NbglA-CbglThm is a balance of the parental enzymes specificities, because they showed clear preference for one of those substrates, whereas the chimera presented the same kcat/Km for both. Hence, the characterization of the chimera activity indicates that the (β/α)4 subdomains folded self-sufficiently and adjusted itself to form a functional active site. In agree, the chimera thermostability also indicates that the (β/α)4 subdomains kept their individual thermal-properties. These observations point to a sufficient thermodynamic stability in the folding and properties of each (β/α)4 half, implying that they were virtually self-contained like true domains. (AU)

• FAPESP's process: 14/16913-8 - Role of the half-barrels subdomains on the stability of beta-glucosidases
• Grantee: Maira Artischeff Frutuoso
• Support Opportunities: Scholarships in Brazil - Doctorate