Creation of active <scp>TIM</scp> barrel enzymes through genetic fusion of half‐barrel domain constructs derived from two distantly related glycosyl hydrolases

During SDS‐PAGE experiments, proteins generally display electrophoretic mobility in keeping with their molecular weights; however, some proteins display anomalies in mobility. Here, we focus attention on the anomalies displayed by the highly acidic ∼110 residues‐long, sequence‐homologous, structurally‐analogous, extracellular domains of human E‐ and N‐cadherin. We report that there is a strong correlation between the acidity of each domain and the degree of the anomaly that it displays. The anomaly is only seen if the ratio of the numbers of negatively‐charged and positively‐charged residues is equal to or greater than the value of 1.50. The degree of the anomaly rises in proportion with this NC:PC ratio. Greater‐than‐expected anomalies are observed for domains containing dense clusters of negatively charged residues. A simple explanation for these observations is that highly acidic domains electrostatically repel SDS. This results in insufficient SDS binding, insufficient electromotive incentive and (consequently) lowered electrophoretic mobility. This explanation is in consonance with the current view that initial stages of SDS‐protein engagement tend to be dominated by electrostatics. We discuss the current anomalies within the broader context of all conceivable explanations for such anomalies.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Understanding anomalous mobility of proteins on SDS‐PAGE with special reference to the highly acidic extracellular domains of human E‐ and N‐cadherins

2019

Self Cite

“…Protein structure and function evolve through sequence changes, substitutions, duplications, insertions, and deletions, including rearrangement or recombination of short fragments as we did here, building on earlier work showing that folded hybrid domains can be generated by shuffling polypeptide segments (36). Based on prior experimental and structural studies, which include specific research in glycoside hydrolases (37,38) and whose results suggest that the common (␤␣) 8 barrel or TIM barrel evolved from an ancestral half-barrel through a series of duplication, fusion, and diversification events (20,39), our work here is in effect accelerated evolution with a particular design goal in mind. The research here is designed to combine the high substrate affinity of a mesophilic enzyme with the thermostability of a thermophilic enzyme to produce more efficient enzymes for specific substrates.…”

Section: Discussionmentioning

confidence: 99%

Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents

Vermaas

et al. 2019

Appl Environ Microbiol

Cellulases from glycoside hydrolase family 5 (GH5) are key endoglucanase enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα)8-barrel structure, where each (βα) module is essential for the enzyme’s stability and activity. Despite their shared topology, the thermostability of GH5 endoglucanase enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on the previously characterized thermophilic GH5 endoglucanase Egl5A from Talaromyces emersonii (TeEgl5A), which has an optimal temperature of 90°C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 from Stegonsporium opalus (SoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optimum (10 and 20°C), the temperature at which the protein lost 50% of its activity (T50) (15 and 19°C), and the melting temperature (Tm) (16.5 and 22.9°C) and extended half-lives (t1/2) (∼240- and 650-fold at 55°C) relative to the values for the mesophilic parent enzyme and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase, Cel5 from Aspergillus niger (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both the SoCel5 and TeEgl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between α2 and α3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parent SoCel5. IMPORTANCE Thermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM barrel fragments, and even fragments from unrelated folds, to generate new structures. However, little research has been done on GH5 endoglucanases. In this study, two GH5 endoglucanases exhibiting TIM barrel structure, SoCel5 and TeEgl5A, with different thermal properties, were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure-guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

“…Protein structure and function evolve through sequence changes, substitutions, duplications, 346 insertions, and deletions, including rearrangement or recombination of short fragments as we 347 did here, building on earlier work showing that folded hybrid domains can be generated by 348 shuffling polypeptide segments (38). Based on prior experimental and structural studies 349 suggesting that the common (βα) 8 reported that a stable and active chimera CelBCelCCA, which showed maximum activity at 359 70°C and was created by fusion the N-half barrel of the thermophilic CelB (maximum 360 activity at 95°C) and C-half barrel of mesophilic CelCCA (maximum activity at 50°C) (40). 361 Furthermore, Numata et al constructed chimeric isopropylmalate dehydrogenases by 362 connecting fragments from a thermophilic and a mesophilic parental enzyme.…”

Section: Discussion 345mentioning

confidence: 96%

“…Most of these hybrids produced inactive 374 proteins, and a few hybrid enzymes had partial or trace activity. Even though the previously 375 mentioned chimera CelBCelCCA displays hyperthermophile-like structural stability, the 376 chimera activity is significant lower than those of the parental enzymes CelB or CelCCA (40). 377…”

Section: Discussion 345mentioning

confidence: 96%

Characterizing Activity and Thermostability of GH5 Cellulase Chimeras from Mesophilic and Thermophilic Parents

Vermaas

et al. 2018

Preprint

23Cellulases from glycoside hydrolase (GH) family 5 are key enzymes in the degradation of 24 diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down 25 biomass. The GH5 family shares a canonical (βα) 8 -barrel structure, where each (βα) module 26 is essential for the enzyme stability and activity. Despite their shared topology, the 27 thermostability of GH5 enzymes can vary significantly, and highly thermostable variants are 28 often sought for industrial applications. Based on a previously characterized thermophilic 29 GH5 cellulase from Talaromyces emersonii (TeEgl5A, with an optimal temperature of 90°C), 30we created ten hybrid enzymes with the mesophilic cellulase from Prosthecium opalus 31 (PoCel5) to determine which elements are responsible for enhanced thermostability. Five of 32 the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited 33 pronounced increases in the temperature optima (10 and 20°C), T 50 (15 and 19°C), T m (16.5 34 and 22.9°C), and extended half life, t 1/2 (~240-and 650-fold at 55°C) relative to the 35 mesophilic parent enzyme, and demonstrated improved catalytic efficiency on selected 36 substrates. The successful hybridization strategies were validated experimentally in another 37 GH5 cellulase from Aspergillus nidulans (AnCel5), which demonstrated a similar increase in 38 thermostability. Based on molecular dynamics simulations (MD) of both PoCel5 and 39TeEgl5A parent enzymes as well as their hybrids, we hypothesize that improved hydrophobic 40 packing of the interface between α 2 and α 3 is the primary mechanism by which the hybrid 41 enzymes increase their thermostability relative to the mesophilic parent PoCel5. 42Thermal stability is an essential property of enzymes in many industrial biotechnological 45 applications, as high temperatures improve bioreactor throughput. Many protein engineering 46 approaches, such as rational design and directed evolution, have been employed to improve 47 the thermal properties of mesophilic enzymes. Structure-based recombination has also been 48 used to fuse TIM-barrel fragments and even fragments from unrelated folds, to generate new 49 structures. However, there are not many research on GH5 cellulases. In this study, two GH5 50 cellulases, which showed TIM-barrel structure, PoCel5 and TeEgl5A with different thermal 51properties were hybridized to study the roles of different (βα) motifs. This work illustrates the 52 role that structure guided recombination can play in helping to identify sequence function 53 relationships within GH5 enzymes by supplementing natural diversity with synthetic 54 diversity. 55 56 Keywords: GH5 Cellulase; (βα) 8 -barrel structure; structure-based recombination; hybrid 57 enzymes; thermostability; 58 59 5 7 conducting structure-guided protein engineering. The cellulase TeEgl5A from T. emersonii (6) 102 exhibits high thermostability, retaining almost all of the activity after incubation at 70°C for 1 103 h, although the structural underpinning for thermal t...