Biology subjects

Evans, R. L.

Publications and source records attributed to Evans, R. L..

8 recordsLinked to original sources

bioRxiv2026

Identifying a cooperative catalytic network for efficient esterase catalysis

Active-site redesign frequently yields modest improvements because residues controlling physical steps like substrate binding and product release lie outside the active site. Efficient catalysis requires a cooperative catalytic network of residues that support both the chemical and physical steps of catalysis. Using ancestral hydroxynitrile lyase HNL1, an /{beta}-hydrolase with poor esterase activity, we tested this framework directly. Matching all active-site residues to a proficient esterase improved KM five-fold but left kcat unchanged, confirming that chemical machinery alone is insufficient. Activity-weighted sequence comparison (SigniSite) across ten homologous HNLs and esterases identified 38 positions disfavoring esterase activity. Experimental refinement yielded a minimal set of fifteen substitutions (HNL1-15) with [~]60-fold higher kcat and [~]400-fold higher kcat/KM. Single-substitution reversion analysis confirmed that all fifteen substitutions are essential and provided evidence for strong cooperativity between them. X-ray crystal structures of HNL1 and HNL1-15 reveal three coordinated structural changes: reshaping the substrate-binding pocket to favor productive ester binding, restoring access to the oxyanion hole, and opening an additional tunnel for product egress and water entry. These changes arise through backbone rearrangements and altered flexibility rather than direct active-site contacts, explaining why the responsible positions escape conservation-based detection. Because cooperativity masks individual contributions, engineering such networks may require step-specific assays -- measuring binding, acylation, or product release directly -- rather than screening composite kcat.

biochemistry↗

bioRxiv2025

Crystal structures of forty- and seventy-one-substitution variants of hydroxynitrile lyase from rubber tree

The /{beta}-hydrolase fold family contains mostly esterases but includes other enzymes such as hydroxynitrile lyase from Hevea brasiliensis (rubber tree, HbHNL). HbHNL shares 44% sequence identity and a Ser-His-Asp catalytic triad with esterase SABP2 (salicylic acid binding protein 2 from Nicotiana tabacum (tobacco)). To identify how large a region within HbHNL influences the positions of the catalytic residues, we created variants where increasingly large regions surrounding the substrate-binding site had identical amino acid sequences to those in SABP2. Variant HNL40 contains 40 mutations (two inserted amino acid residues, 38 substitutions), shares 59% sequence identity with SABP2, and is identical in sequence to SABP2 within 10 [A] of the substrate-binding site. Variant HNL71 contains 31 additional substitutions for a total of 71 changes (two insertions, 69 substitutions) and shares 71% sequence identity with SABP2. The sequences within 14 [A] of the substrate-binding site are identical in SABP2 and HNL71. The crystal structures of HNL40 and HNL71 show that the positions of main chain C[a] atoms move from their positions in HbHNL to more closely match those in SABP2 (RMSD = 0.51 [A] over 235 C[a] atoms for HNL40, 0.41 [A] over 219 C[a] atoms for HNL71) and even more closely in the region within 10 [A] of the substrate-binding site (RMSD = 0.38 [A] over 58 C[a] atoms for HNL40, 0.28 [A] over 53 C[a] atoms for HNL71). The pattern of tunnels in HNL40 and HNL71 are similar to each other and intermediate between the pattern in HbHNL and SABP2. SynopsisVariants HNL40 and HNL71 of hydroxynitrile lyase from Hevea brasiliensis contain 40 and 71 mutations, respectively, to make regions surrounding the substrate-binding site identical in sequence to esterase SABP2. X-ray structures reveal increasing similarities to SABP2 in HNL40 and HNL71. PDB reference: hydroxynitrile lyase from Hevea brasiliensis with forty mutations, 8SNI, hydroxynitrile lyase from Hevea brasiliensis with seventy-one mutations, 9CLR

biochemistry↗

bioRxiv2025

AI, citizen science, and the 2024 eclipse emphasize the importance of light for bird behavior

On April 8th 2024, a total solar eclipse disrupted light-dark cycles for North American birds during the lead-up to spring reproduction. Compiling over 10,000 community observations and AI analyses of nearly 100,000 vocalizations, we found that bird behavior was significantly affected by these few minutes of unexpected afternoon darkness. More than half of wild bird species changed their biological rhythms, with many producing a dawn chorus in the aftermath of the eclipse. This natural experiment demonstrates the power of technology-enabled and public science projects to understand our natural world. Further, it underscores the power of light in structuring animal behavior: even when night lasts for just four minutes, robust behavioral changes ensue.

animal behavior and cognition↗

bioRxiv2023

Designing Efficient Enzymes: Eight Predicted Mutations Convert a Hydroxynitrile Lyase into an Efficient Esterase

Hydroxynitrile lyase from rubber tree (HbHNL) shares 45% identical amino acid residues with the homologous esterase from tobacco, SABP2, but the two enzymes catalyze different reactions. The x-ray structures reveal a serine-histidine-aspartate catalytic triad in both enzymes along with several differing amino acid residues within the active site. Previous exchange of three amino acid residues in the active site of HbHNL with the corresponding amino acid residue in SABP2 (T11G-E79H-K236M) created variant HNL3, which showed low esterase activity toward p-nitrophenyl acetate. Further structure comparison reveals additional differences surrounding the active site. HbHNL contains an improperly positioned oxyanion hole residue and differing solvation of the catalytic aspartate. We hypothesized that correcting these structural differences would impart good esterase activity on the corresponding HbHNL variant. To predict the amino acid substitutions needed to correct the structure, we calculated shortest path maps for both HbHNL and SABP2, which reveal correlated movements of amino acids in the two enzymes. Replacing four amino acid residues (C81L-N104T-V106F-G176S) whose movements are connected to the movements of the catalytic residues yielded variant HNL7TV (stabilizing substitution H103V was also added), which showed an esterase catalytic efficiency comparable to that of SABP2. The x-ray structure of an intermediate variant, HNL6V, showed an altered solvation of the catalytic aspartate and a partially corrected oxyanion hole. This dramatic increase in catalytic efficiency demonstrates the ability of shortest path maps to predict which residues outside the active site contribute to catalytic activity.

bioengineering↗

bioRxiv2022

Structure and function of a Notch-like proteolytic switch domain in the extracellular matrix receptor dystroglycan

The dystrophin glycoprotein complex (DGC), anchored by the transmembrane protein dystroglycan, functions to mechanically link the extracellular matrix to the actin cytoskeleton to drive critical aspects of development and adult homeostasis. Breaking this connection via mutation of the actin adaptor protein dystrophin or impaired glycosylation of dystroglycan are strongly associated with diseases such as Muscular Dystrophy, yet cleavage of the dystroglycan protein by matrix metalloproteinases (MMPs) remains an understudied mechanism to disrupt the DGC. We solved X-ray structures of the membrane-adjacent domain of dystroglycan to understand the molecular underpinnings of dystroglycan MMP cleavage regulation. Dystroglycan proteolysis occurs within the versatile SEAL domain, which supports proteolysis in diverse receptors to facilitate mechanotransduction, protection of cell membranes, and even viral entry. The structure reveals a c-terminal extension of the SEAL domain that buries the MMP cleavage site by packing into a hydrophobic pocket, a unique mechanism of MMP cleavage regulation. We further demonstrate that structure-guided and disease-associated mutations disrupt proteolytic regulation using a new cell-surface proteolysis assay. Finally, we find that disruption of proteolysis leads to altered cellular mechanics and migration using high-throughput DNA tension probe and wound healing assays. These findings highlight that disrupted proteolysis is a relevant mechanism for "breaking" the DGC link to contribute to disease pathogenesis and may offer new therapeutic avenues for dystroglycanopathies.

biochemistry↗

bioRxiv2020

Molecular underpinnings of ssDNA specificity by Rep HUH endonucleases and implications for HUH-tag multiplexing and engineering

Replication initiator proteins (Reps) from the HUH-endonuclease superfamily process specific single-stranded DNA (ssDNA) sequences to initiate rolling circle/hairpin replication in viruses, such as crop ravaging geminiviruses and human disease causing parvoviruses. In biotechnology contexts, Reps are the basis for HUH-tag bioconjugation and a critical adeno-associated virus genome integration tool. We solved the first co-crystal structures of Reps complexed to ssDNA, revealing a key motif for conferring sequence specificity and for anchoring a bent DNA architecture. In combination, we developed a deep sequencing cleavage assay, termed HUH-seq, to interrogate subtleties in Rep specificity and demonstrate how differences can be exploited for multiplexed HUH-tagging. Together, our insights allowed engineering of only four amino acids in a Rep chimera to predictably alter sequence specificity. These results have important implications for modulating viral infections, developing Rep-based genomic integration tools, and enabling massively parallel HUH-tag barcoding and bioconjugation applications.

biophysics↗

bioRxiv2020

Student-focused development of a next-generation centrifuge force microscope

Advanced biological molecule force probing methods such as atomic force microscopy and optical tweezers used to quantify forces at the single-molecule level are expensive and require extensive training and technical knowledge. However, the technologies underlying a centrifuge force microscope (CFM) are relatively straight forward, allowing for construction by labs with relatively low budgets and minimal training. Design ideas from previously constructed CFMs served as a guide in the development of this CFM. There were two primary goals: first, to develop an inexpensive, functional CFM using off-the-shelf and 3D printed parts; and second, to do so in the context of providing an educational experience for a broad range of students. The team included high school students and undergraduates from local high schools, the University of Minnesota, and other local higher education institutions. This project created an environment for student-focused development of the CFM that fostered active learning, individual ownership, as well as excellence in research. The instrument discussed herein represents a fully functional CFM designed and built by a postdoctoral researcher and a graduate student who together mentored several high school and undergraduate students. STATEMENT OF SIGNIFICANCEThe presented centrifuge force microscope (CFM) builds on features of existing designs specifically engineered for probing macromolecular force interactions at the single-molecule level. In the coming years, more versatile and modular CFM designs will be utilized in the force spectroscopy field, and the presented design is a step in that direction. In addition to constructing a functional instrument, true student ownership of the project design was equally an end goal. Students from high school through graduate school were included, and the project was structured so that everyone was seen as peers. This active learning project allowed students to acquire scientific concepts and techniques and apply them to real-life situations.

biophysics↗

bioRxiv2020

Larger active site in an ancestral hydroxynitrile lyase increases catalytically promiscuous esterase activity

Hydroxynitrile lyases (HNLs) belonging to the /{beta}-hydrolase-fold superfamily evolved from esterases approximately 100 million years ago. Reconstruction of an ancestral hydroxynitrile lyase in the /{beta}-hydrolase fold superfamily yielded a catalytically active hydroxynitrile lyase, HNL1. Several properties of HNL1 differ from the modern HNL from rubber tree (HbHNL). HNL1 favors larger substrates as compared to HbHNL, is two-fold more catalytically promiscuous for ester hydrolysis (p-nitrophenyl acetate) as compared to mandelonitrile cleavage, and resists irreversible heat inactivation to 35 {degrees}C higher than for HbHNL. We hypothesized that the x-ray crystal structure of HNL1 may reveal the molecular basis for the differences in these properties. The x-ray crystal structure solved to 1.96-[A] resolution shows the expected /{beta}-hydrolase fold, but a 60% larger active site as compared to HbHNL. This larger active site echoes its evolution from esterases since related esterase SABP2 from tobacco also has a 38% larger active site than HbHNL. The larger active site in HNL1 likely accounts for its ability to accept larger hydroxynitrile substrates. Site-directed mutagenesis of HbHNL to expand the active site increased its promiscuous esterase activity 50-fold, consistent with the larger active site in HNL1 being the primary cause of its promiscuous esterase activity. Urea-induced unfolding of HNL1 indicates that it unfolds less completely than HbHNL (m-value = 0.63 for HNL1 vs 0.93 kcal/ mol{middle dot}M for HbHNL), which may account for the ability of HNL1 to better resist irreversible inactivation upon heating. The structure of HNL1 shows changes in hydrogen bond networks that may stabilize regions of the folded structure.

biochemistry↗