Structural analysis and functional evaluation of the disordered ß–hexosyltransferase region from Hamamotoa (Sporobolomyces) singularis

Dagher, Suzanne F.; Vaishnav, Asmita; Stanley, Christopher B.; Meilleur, Flora; Edwards, Brian P.; Bruno-Bárcena, José M.

doi:10.3389/fbioe.2023.1291245

Title: Structural analysis and functional evaluation of the disordered ß–hexosyltransferase region from Hamamotoa (Sporobolomyces) singularis

Journal Article · 14 December 2023 · Frontiers in Bioengineering and Biotechnology

DOI: https://doi.org/10.3389/fbioe.2023.1291245 · OSTI ID:2281169

Dagher, Suzanne F. ^[1]; Vaishnav, Asmita ^[2]; Stanley, Christopher B. ^[3]; Meilleur, Flora ^[4]; Edwards, Brian P. ^[2]; Bruno-Bárcena, José M. ^[1]

North Carolina State University, Raleigh, NC (United States)
Wayne State University, Detroit, MI (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); North Carolina State University, Raleigh, NC (United States)

Hamamotoa (Sporobolomyces) singularis codes for an industrially important membrane bound ß-hexosyltransferase (BHT), (BglA, UniprotKB: Q564N5) that has applications in the production of natural fibers such as galacto-oligosaccharides (GOS) and natural sugars found in human milk. When heterologously expressed by Komagataella phaffii GS115, BHT is found both membrane bound and soluble secreted into the culture medium. In silico structural predictions and crystal structures support a glycosylated homodimeric enzyme and the presence of an intrinsically disordered region (IDR) with membrane binding potential within its novel N-terminal region (1–110 amino acids). Additional in silico analysis showed that the IDR may not be essential for stable homodimerization. Thus, we performed progressive deletion analyses targeting segments within the suspected disordered region, to determine the N-terminal disorder region’s impact on the ratio of membrane-bound to secreted soluble enzyme and its contribution to enzyme activity. The ratio of the soluble secreted to membrane-bound enzyme shifted from 40% to 53% after the disordered N-terminal region was completely removed, while the specific activity was unaffected. Furthermore, functional analysis of each glycosylation site found within the C-terminal domain revealed reduced total secreted protein activity by 58%–97% in both the presence and absence of the IDR, indicating that glycosylation at all four locations is required by the host for the secretion of active enzyme and independent of the removed disordered N-terminal region. Overall, the data provides evidence that the disordered region only partially influences the secretion and membrane localization of BHT.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States); Argonne National Laboratory (ANL), Argonne, IL (United States); North Carolina State University, Raleigh, NC (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES). Scientific User Facilities (SUF); Michigan Economic Development Corporation (MEDC); Michigan Technology Tri-Corridor

Grant/Contract Number:: AC05-00OR22725; AC02-06CH11357; 085P1000817; 2018-2092

OSTI ID:: 2281169

Journal Information:: Frontiers in Bioengineering and Biotechnology, Vol. 11, Issue 1; ISSN 2296-4185

Publisher:: Frontiers Media S.A.Copyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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Hamamotoa singularis

Title: Structural analysis and functional evaluation of the disordered ß–hexosyltransferase region from Hamamotoa (Sporobolomyces) singularis

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