Kang, L.-W. | bioRxiv Science

bioRxiv2021

Temperature-responsive structural reversibility of FGF21 and structure-based design of its variant with enhanced potency

Fibroblast growth factor 21 (FGF21) has pharmaceutical potential against obesity-related metabolic disorders, including non-alcoholic fatty liver disease. Since thermal stability is a desirable factor for therapeutic proteins, we investigated the thermal behavior of human FGF21. FGF21 remained soluble after heating; thus, we examined its temperature-induced structural changes using circular dichroism (CD). FGF21 showed inter-convertible temperature-specific CD spectra. The CD spectrum at 100 {degrees}C returned to that at 20 {degrees}C when the heated FGF21 solution was cooled. Through loop swapping, the connecting loop between {beta}10 and {beta}12 in FGF21 was revealed to be associated with the unique thermal behavior of FGF21. According to in vitro cell-based assays and model high-fat diet (HFD)-induced obesity studies, heated FGF21 maintained biological activities that were comparable to those of non-heated and commercial FGF21s. Based on sequence comparison and structural analysis, five point-mutations were introduced into FGF21. Compared with the wild type, the heated FGF21 variant displayed improved therapeutic potential in terms of body weight loss, the levels of hepatic triglycerides and lipids, and the degree of vacuolization of liver in HFD-fed mice.

biochemistry↗

bioRxiv2019

Dual activity of PNGM-1, a metallo-β-lactamase and tRNase Z, pinpoints the evolutionary origin of subclass B3 metallo-β-lactamases

Antibiotic resistance is a steadily increasing global problem which could lead to a fundamental upheaval in clinical care with the potential to return us to the pre-antibiotic era1-4. The production of {beta}-lactamases, a group of enzymes that confer antibiotic resistance in Gram-negative bacteria, is now one of the major barriers in treating Gram-negative infections5. {beta}-Lactamases are classified according to their catalytic mechanisms into serine {beta}-lactamases and metallo-{beta}-lactamases6,7. There are functional and structural similarities between serine {beta}-lactamases and penicillin-binding proteins, and so serine {beta}-lactamases are thought to have evolved from a penicillin-binding protein7,8. Given the functional and structural differences between serine {beta}-lactamases and metallo-{beta}-lactamases, metallo-{beta}-lactamases are thought to have evolved from a protein other than a penicillin-binding protein, but to date this ancestor remains unknown8-11. We discovered PNGM-1, the first subclass B3 metallo-{beta}-lactamase, in deep-sea sediments that predate the antibiotic era12. Here we discover the dual activity of PNGM-1, pinpointing the evolutionary origin of subclass B3 metallo-{beta}-lactamases. Phylogenetic analysis suggested that PNGM-1 could yield insights into the evolutionary origin of subclass B3 metallo-{beta}-lactamases. We reveal the structural similarities between tRNase Zs and PNGM-1, which prompted us to investigate their evolutionary relationship and the possibility of them possessing dual enzymatic activities. We demonstrate that PNGM-1 has dual activity with both true metallo-{beta}-lactamase and tRNase Z activity, suggesting that PNGM-1 is thought to have evolved from a tRNase Z. We also show kinetic and structural comparisons between PNGM-1 and other proteins including subclass B3 metallo-{beta}-lactamases and tRNase Zs. These comparisons revealed that the B3 metallo-{beta}-lactamase activity of PNGM-1 is a promiscuous activity and subclass B3 metallo-{beta}-lactamases are thought to have evolved through PNGM-1 activity. Our work provides a foundation for the evolution of tRNase Z into subclass B3 metallo-{beta}-lactamases through the dual activity of PNGM-1.

microbiology↗