Biology subjects

Mandl, S.

Publications and source records attributed to Mandl, S..

3 recordsLinked to original sources

bioRxiv2025

Boosting reversible photocontrol of a photoxenase by an engineered conformational shift

Our study successfully explores strategies to effectively improve the photocontrol efficiency of light-sensitive enzymes, dubbed photoxenases, with photoswitchable unnatural amino acids (UAAs). The engineering of photoxenases is a versatile method for the reversible photocontrol in various applications. To boost the photocontrol of an established allosteric and heterodimeric photoxenase based on imidazole glycerol phosphate synthase, we turned from an ineffective tuning of the UAA photochemistry to a semi-rational enzyme design. Remarkably, mutations at the catalytically important heterodimer interface increased the light-regulation factor (LRF) for the kcat up to ~100 with near-quantitative reversibility. Steady-state kinetic investigations combined with computationally determined correlation-based Shortest-Path-Map analysis and conformational landscapes revealed how photocontrol was altered in the two best hits. The LRF(kcat) correlated with a shift of a conformational equilibrium between an active and inactive population at the targeted active site and a tuned population productivity upon irradiation. While the overall reduced kcat values originated from a rewiring of the allosteric signal transmission, the increased LRF(kcat) resulted from a change in i) the size of the conformational shift, ii) the population productivity, and iii) the conformational heterogeneity. With this, our findings provide initial guidelines to boost photocontrol and underscore the power of photoxenase engineering.

biochemistry↗

bioRxiv2024

Promoting enzyme catalysis via azobenzene facilitated vibrational energy transfer

Azobenzene is a widely recognized tool for achieving artificial spatiotemporal control of enzyme activity using light. Photocontrol reversibility is typically based on photostationary states with varying E and Z isomer compositions achieved through irradiation at specific wavelengths. Here, we report an alternative mechanism for azobenzene based enzyme regulation, discovered through simultaneous irradiation with two wavelengths. Using two engineered variants of imidazole glycerol phosphate synthase, in which azobenzene was incorporated as an unnatural amino acid to enable reversible control under monochromatic irradiation, we uncovered unique behavior under dichromatic irradiation. Notably, a distinct spectroscopic signal from the azobenzene moiety emerged during simultaneous irradiation at 365 nm and 420 nm and vanished upon return to the dark. Intriguingly, dichromatic irradiation triggered a reproducible two-fold increase in catalytic activity and an instantaneous return to baseline activity in the dark for one variant. In contrast, the other variant and the wild-type enzyme maintained their baseline activity under the same conditions. These findings reveal an unexplored avenue for azobenzene photoswitching, offering a novel approach to photocontrol with potential applications in sequentially regulating multiple enzymes, especially when combined with monochromatic irradiation strategies.

biophysics↗

bioRxiv2018

Fermentation characteristics of mead and wine generated by yeasts isolated from beehives of two Austrian regions

Mead is a traditional alcoholic beverage that is produced by fermentation of diluted honey. The mead quality is primarily influenced by the honey variety, although the yeast microflora as the main catalyst of alcoholic fermentation also plays a significant role in the organoleptic and chemical quality of the final product. The impact of the indigenous honey associated-yeasts on the mead properties has scarcely been investigated. To fill this gap the main objective of this work was to assess the metabolic properties of the yeasts isolated from honey and pollen from beehives of northeast Austria.\n\nThe biodiversity was low and only two yeast species were identified, Zygosaccharomyces rouxii and Candida apicola. The fermentation potentials of these yeasts were estimated in two media, grape juice (since yeasts isolated from honey may be useful for sweet wine production) and diluted honey of similar sugar concentration, and compared with those of the reference strains Saccharomyces cerevisiae; S. uvarum and S. eubayanus. Depending on the fermentation substrate, yeasts differed with respect to their metabolic power, fermentation rate, sugar utilization and production of glycerol and organic acids. During mead fermentation Saccharomyces species showed the highest metabolic turnover, while the fermentation rate did not differ significantly. Addition of assimilable nitrogen to the diluted honey enhanced fermentation rate of S. cerevisiae, but not of the other species. Fermentation of grape juice occurred much faster than that of diluted honey and differences between yeasts were more pronounced. The S. cerevisiae commercial wine strain, adapted to high alcohol concentrations, and S. eubayanus outperformed the others, S. uvarum was comparable with Z. rouxii, while C. apicola had the lowest fermentation rate. Fructophily of Z. rouxii and to a lesser degree of C. apicola was observed in both media. An increased production of glycerol was achieved by S. eubayanus in both media and by C. apicola during the fermentation of honey must. A commercial S. cerevisiae strain, S. eubayanus and Z. rouxii were able to metabolize malic acid in wine. In mead, the S. eubayanus and S. uvarum yeasts showed the tendency of increasing the level of malic acid. Aroma profile depended profoundly on yeast species. This study demonstrates that the composition and complexity of the fermentation substrate determines the activity and the final metabolic outcomes of the studied yeasts.

microbiology↗