The Critical Role of Partially Exposed N-Terminal Valine Residue in Stabilizing GH10 Xylanase from Bacillus sp.NG-27 under Poly-Extreme Conditions

Abstract

BackgroundUnderstanding the mechanisms that govern protein stability under poly-extreme conditions continues to be a major challenge. Xylanase (BSX) from Bacillus sp. NG-27, which has a TIM-barrel structure, shows optimum activity at high temperature and alkaline pH, and is resistant to denaturation by SDS and degradation by proteinase K. A comparative circular dichroism analysis was performed on native BSX and a recombinant BSX (R-BSX) with just one additional methionine resulting from the start codon. The results of this analysis revealed the role of the partially exposed N-terminus in the unfolding of BSX in response to an increase in temperature.MethodologyWe investigated the poly-extremophilicity of BSX to deduce the structural features responsible for its stability under one set of conditions, in order to gain information about its stability in other extreme conditions. To systematically address the role of the partially exposed N-terminus in BSX stability, a series of mutants was generated in which the first hydrophobic residue, valine (Val1), was either deleted or substituted with various amino acids. Each mutant was subsequently analyzed for its thermal, SDS and proteinase K stability in comparison to native BSX.ConclusionsA single conversion of Val1 to glycine (Gly) changed R-BSX from being thermo- and alkali- stable and proteinase K and SDS resistant, to being thermolabile and proteinase K-, alkali- and SDS- sensitive. This result provided insight into the structure-function relationships of BSX under poly-extreme conditions. Molecular, biochemical and structural data revealed that the poly-extremophilicity of BSX is governed by a partially exposed N-terminus through hydrophobic interactions. Such hitherto unidentified N-terminal hydrophobic interactions may play a similar role in other proteins, especially those with TIM-barrel structures. The results of the present study are therefore of major significance for protein folding and protein engineering.

Structural insights into N‐terminal to C‐terminal interactions and implications for thermostability of a (β/α)8‐triosephosphate isomerase barrel enzyme

“…RBSX and its mutants were found to unfold in an irreversible manner, and the apparent melting temperature ( T m ) was calculated for all protein samples with a constant temperature slope of 60 °C·h −1 . It was observed that the V1G and V1A mutations decreased the stability of the protein by 12 °C and 2 °C, respectively, whereas the V1F and V1D mutations did not significantly change its thermostability, as compared with RBSX . On the other hand, the V1L mutation markedly enhanced the thermostability of RBSX from 70 °C to 75 °C without compromising its catalytic activity, and resulted in higher cooperativity in the thermal unfolding transition .…”

“…In an earlier study from our group, different extreme N‐terminus mutants of RBSX were generated in which the first residue, Val1, was replaced with various amino acids, and each mutant was subsequently analyzed for its thermal stability at high temperatures. The thermal stabilities of RBSX and the mutant proteins were determined by CD measurements at a range of temperatures . RBSX and its mutants were found to unfold in an irreversible manner, and the apparent melting temperature ( T m ) was calculated for all protein samples with a constant temperature slope of 60 °C·h −1 .…”

“…It was observed that the V1G and V1A mutations decreased the stability of the protein by 12°C and 2°C, respectively, whereas the V1F and V1D mutations did not significantly change its thermostability, as compared with RBSX [16]. On the other hand, the V1L mutation markedly enhanced the thermostability of RBSX from 70°C to 75°C without compromising its catalytic activity, and resulted in higher cooperativity in the thermal unfolding transition [16]. However, at present, structural details are not available for any mutants except the V1A and V1L mutants.…”

“…The thermal stabilities of RBSX and the mutant proteins were determined by CD measurements at a range of temperatures [16]. RBSX and its mutants were found to unfold in an irreversible manner, and the apparent melting temperature (T m ) was calculated for all protein samples with a constant temperature slope of 60°CÁh À1 .…”

“…Earlier studies of recombinant BSX (RBSX) showed that just a single extreme N‐terminus mutation, V1L, although not located in any secondary structural element (SSE) (helices or β‐sheets), markedly enhanced RBSX stability from 70 °C to 75 °C without loss of catalytic activity . In contrast, the V1A mutation at the same position decreased the stability of the enzyme from 70 °C to 68 °C.…”