Key amino acid residues govern the substrate selectivity of the transporter Xltr1p from Trichoderma reesei for glucose, mannose, and galactose

Abstract

This research identified four amino acid residues (Leu174, Asn297, Tyr301, and Gln291) that contribute to substrate recognition by the high-affinity glucose transporter Xltr1p from Trichoderma reesei. Potential hotspots affecting substrate specificity were selected through homology modeling, evolutionary conservation analyses, and substrate-docking modeling of Xltr1p. Variants carrying mutations at these hotspots were subsequently obtained via in silico screening. Replacement of Leu174 or Asn297 in Xltr1p with alanine resulted in loss of hexose transport activity, indicating that Leu174 and Asn297 play essential roles in hexose transport. The Y301W variant exhibited accelerated mannose transport, but lost galactose transport capacity, and mutation of Gln291 to alanine greatly accelerated mannose transport. These results suggest that amino acids located in transmembrane α-helix 7 (Asn297, Tyr301, and Gln291) play critical roles in substrate recognition by the hexose transporter Xltr1p. Our results will help expand the potential applications of this transporter and provide insights into the mechanisms underlying its function and specificity.

Keywords

Hexose transporter / Glucose / Galactose / Mannose / Transmembrane α-helix 7 / Sugar / ransport / Computer-aided screening

Data Availability Statement

The original/source data and resources are available from the correspondence author Xu Fang ([e-mail]) on request.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Wei Ma: Writing - review & editing, Writing - original draft, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization. Shiyu Yuan: Supervision, Formal analysis. Zixian Wang: Supervision, Formal analysis. Kangle Niu: Supervision, Methodology. Fengyi Li: Writing - original draft, Supervision. Lulu Liu: Supervision, Formal analysis. Lijuan Han: Supervision, Methodology. Xu Fang: Writing - review & editing, Writing - original draft, Supervision, Project administration, Methodology, Funding acquisition, Conceptualization.

Acknowledgments

The authors would like to thank Dr. Xiangmei Ren from State Key Laboratory of Microbial Technology of Shandong University for help and guidance in the high-performance liquid chromatography. We thank Prof. Dr Eckhard Boles from the Institutfür Molekulare Biowissenschaf-ten Goethe-Universität Frankfurt for kindly providing the EBY.VW4000 strain. This work was supported by National Key R&D Program of China (No. 2018YFA0901700), National Natural Science Foundation of China (No. 32271526).

Chemical compounds studied in this article

d-Glucose (PubChem CID:5793), d-Mannose (PubChem CID:18950), d-Galactose (PubChem CID:6036).

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