Propeptide-Mediated Allosteric Regulation of Xylanase Xyl-1: An Integrated Experimental and Computational Analysis

Abstract

Most GH11 family endo-β-1,4-xylanases contain a propeptide region linked to the N-terminal region. The mechanistic basis of this region harboring key regulation information for enzyme function, however, remains poorly understood. We reported an investigation on the allosteric regulation mechanism of the propeptide based on biochemical characterization, molecular dynamics simulations, and evolutionary analysis. We discovered that the mutant of truncated propeptide shows a remarkably increased thermal stability (melting temperature increased by 11.5 °C) and catalytic efficiency (1.7-fold k cat /K m value of wild type). Molecular dynamics simulations reveal that long-range fluctuations in the propeptide lead to a conformational perturbation in the catalytic pocket and the thumb region. The probability of sampling the active conformation during the glycosylation step is reduced (i.e., catalytic efficiency). In-depth sequence analysis indicates that the propeptide has a strong plasticity and degeneration trend, and propeptide truncation experiments of the homologous enzyme XynB validated the feasibility of the truncation strategy. This work reveals the role of GH11 family propeptides in functional regulation and provides a straightforward and practical method to increase the robustness of GH11 family xylanases.

Noteworthy Consensus Effects of D/E Residues in Umami Peptides Used for Designing the Novel Umami Peptides

“…29 Distal allosteric sites were successfully identified in the evolution of allosteric activity. 50,51 In this study, the SPM method was used to analyze the force conduction during the interaction between peptides and proteins, and it was found that the SPM pathways were consistent with the D/E consensus effector group (Figure 6B). This consistency in contact transmission aligned with the inheritance underlying the consensus effect.…”

Unleashing the Power of Evolution in Xylanase Engineering: Investigating the Role of Distal Mutation Regulation

“…The targeted gene encoding xylanase M4 (D30P/A33Q/K117Q/A167H) was linked to vector pET-32a. Given that E175 is a catalytic amino acid, the E175A mutant on M4 was subsequently developed through site-directed mutagenesis, with a primer of 5′-CAACCGCAGGTTATCAGAGCAGCGGTAG-3′ . Both recombinant plasmids, pET-32a-M4 and pET-32a-E175A, were transformed into E.…”

“…Given that E175 is a catalytic amino acid, the E175A mutant on M4 was subsequently developed through site-directed mutagenesis, with a primer of 5′-CAACCGCAGGTTATCAGAGCAGCGGTAG-3′. 18 Both recombinant plasmids, pET-32a-M4 and pET-32a-E175A, were transformed into E. coli BL21 (DE3) for protein expression and purification, utilizing the methodology described in the "Sitedirected Mutagenesis, Recombinant Protein Expression and Purification" section in the Supporting Information. The purified protein was concentrated to 40 mg/mL in 25 mM Tris, pH 7.5, and 150 mM NaCl and stored at −80 °C for crystallization.…”

“…17 In our previous research, we investigated the enzymatic properties of Xyl-1, a xylanase from the GH11 family that was isolated from Aspergillus terreus, and conducted studies on its application in textile processing. 18,19 This study utilizes xylanase Xyl-1 as a model to delve deeper into the evolution-catalysis relationship and the selection of candidate mutations through an analysis from PSSM. We assess the differences in regional specificity among mutation sites.…”

“…Compared with other families of xylanases, the low molecular size of xylanases belonging to the GH11 family, together with their strong substrate selectivity and catalytic efficiency, make these enzymes a suitable candidate for industrial use . In our previous research, we investigated the enzymatic properties of Xyl-1, a xylanase from the GH11 family that was isolated from Aspergillus terreus, and conducted studies on its application in textile processing. , This study utilizes xylanase Xyl-1 as a model to delve deeper into the evolution-catalysis relationship and the selection of candidate mutations through an analysis from PSSM. We assess the differences in regional specificity among mutation sites.…”