Effect of active-site aromatic residues Tyr or Phe on activity and stability of glucose 6-phosphate dehydrogenase from psychrophilic Arctic bacterium Sphingomonas sp.

Tran, Kiet N.; Jang, Sei‐Heon; Lee, ChangWoo

doi:10.1016/j.bbapap.2020.140543

Cited by 9 publications

(12 citation statements)

References 40 publications

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“…Psychrophilic esterases prefer tyrosine for conformational flexibility, whereas psychrotrophic or mesophilic esterases prefer the more hydrophobic tryptophan for structural stability [ 32 , 33 , 35 ]. On the other hand, glucose 6-phosphate dehydrogenase (G6PD) isozymes choose tyrosine or phenylalanine in the substrate-binding pocket depending on the metabolic pathways [ 34 ]. G6PD1, which is involved in the Entner-Doudoroff pathway, prefers tyrosine, which can form a hydrogen bond with the phosphate group of glucose 6-phosphate, for its enzymatic activity [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

“…On the other hand, glucose 6-phosphate dehydrogenase (G6PD) isozymes choose tyrosine or phenylalanine in the substrate-binding pocket depending on the metabolic pathways [ 34 ]. G6PD1, which is involved in the Entner-Doudoroff pathway, prefers tyrosine, which can form a hydrogen bond with the phosphate group of glucose 6-phosphate, for its enzymatic activity [ 34 ]. By contrast, G6PD2, which is involved in the oxidative pentose phosphate pathway, prefers purely hydrophobic phenylalanine for its thermal stability [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

“…G6PD1, which is involved in the Entner-Doudoroff pathway, prefers tyrosine, which can form a hydrogen bond with the phosphate group of glucose 6-phosphate, for its enzymatic activity [ 34 ]. By contrast, G6PD2, which is involved in the oxidative pentose phosphate pathway, prefers purely hydrophobic phenylalanine for its thermal stability [ 34 ].…”

Section: Discussionmentioning

confidence: 99%

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Essential roles of buried phenylalanine in the structural stability of thioredoxin from a psychrophilic Arctic bacterium Sphingomonas sp.

et al. 2021

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Thioredoxin (Trx), a small redox protein, exhibits thermal stability at high temperatures regardless of its origin, including psychrophiles. Trxs have a common structure consisting of the central β-sheet flanked by an aliphatic cluster on one side and an aromatic cluster on the other side. Although the roles of aromatic amino acids in the folding and stability of proteins have been studied extensively, the contributions of aromatic residues to the stability and function of Trx, particularly Trxs from cold-adapted organisms, have not been fully elucidated. This study examined the roles of aromatic amino acids in the aromatic cluster of a Trx from the psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621 (SpTrx). The aromatic cluster of SpTrx was comprised of W11, F26, F69, and F80, in which F26 at the β2 terminus was buried inside. The substitution of tyrosine for F26 changed the SpTrx conformation substantially compared to that of F69 and F80. Further biochemical and spectroscopic investigations on F26 showed that the F26Y, F26W, and F26A mutants resulted in structural instability of SpTrx in both urea- and temperature-induced unfolding and lower insulin reduction activities. The Trx reductase (SpTR) showed lower catalytic efficiencies against F26 mutants compared to the wild-type SpTrx. These results suggest that buried F26 is essential for maintaining the active-site conformation of SpTrx as an oxidoreductase and its structural stability for interactions with SpTR at colder temperatures.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Essential roles of buried phenylalanine in the structural stability of thioredoxin from a psychrophilic Arctic bacterium Sphingomonas sp.

et al. 2021

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“…Therefore, contact surfaces were probably formed in the protein that was favorable for bonding, thereby increasing the probability of direct contact with the substrates or stabilizing the structure. 37,38 Meanwhile, they also exhibited a slightly higher protein yield than the other mutants. This might reinforce the earlier view that the structures of these four mutants apparently became more stable and flexible thereby reducing damage during protein expression.…”

Section: Resultsmentioning

confidence: 95%

“…an aromatic ring in phenylalanine and an imidazole group in histidine). Therefore, contact surfaces were probably formed in the protein that was favorable for bonding, thereby increasing the probability of direct contact with the substrates or stabilizing the structure 37,38 . Meanwhile, they also exhibited a slightly higher protein yield than the other mutants.…”

Section: Resultsmentioning

confidence: 99%

Enhancing lactose recognition of a key enzyme in 2′‐fucosyllactose synthesis: α‐1,2‐fucosyltransferase

et al. 2022

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BACKGROUND: 2 0 -Fucosyllactose, a representative oligosaccharide in human milk, is an emerging and promising food and pharmaceutical ingredient due to its powerful health benefits, such as participating in immune regulation, regulation of intestinal flora, etc. To enable economically viable production of 2 0 -fucosyllactose, different biosynthesis strategies using precursors and pathway enzymes have been developed. The ⊍-1,2-fucosyltransferases are an essential part involved in these strategies, but their strict substrate selectivity and unsatisfactory substrate tolerance are one of the key roadblocks limiting biosynthesis.RESULTS: To tackle this issue, a semi-rational manipulation combining computer-aided designing and screening with biochemical experiments were adopted. The mutant had a 100-fold increase in catalytic efficiency compared to the wild-type. The highest 2 0 -fucosyllactose yield was up to 0.65 mol mol −1 lactose with a productivity of 2.56 g mL −1 h −1 performed by enzymatic catalysis in vitro. Further analysis revealed that the interactions between the mutant and substrates were reduced. The crucial contributions of wild-type and mutant to substrate recognition ability were closely related to their distinct phylotypes in terms of amino acid preference. CONCLUSION: It is envisioned that the engineered ⊍-1,2-fucosyltransferase could be harnessed to relieve constraints imposed on the bioproduction of 2 0 -fucosyllactose and lay a theoretical foundation for elucidating the substrate recognition mechanisms of fucosyltransferases.

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Tyr76 is essential for the cold adaptation of a class II glutaredoxin 4 with a heat‐labile structure from the Arctic bacteriumSphingomonassp.

et al. 2023

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Glutaredoxins (Grxs) are small proteins that share a well‐conserved thioredoxin (Trx)‐fold and participate in many biological processes. This study examined the cold adaptation mechanism of a Fe‐S cluster binding class II Grx4 (SpGrx4) from the psychrophilic Arctic bacterium Sphingomonas sp. PAMC 26621. Three polar residues close to the cis‐proline residue (P73) of SpGrx4 form a hydrogen bond network (Q74–S67–Y76) with the cis‐proline loop main chain. The hydroxyl group of S67 or Y76 or both is replaced in similar Grxs depending on the temperature of the habitat. Mutants with reduced hydrogen bonds (S67A, Q74A, Y76F, and S67A/Y76W) were more susceptible to urea‐induced unfolding and more flexible than the wild‐type (WT). By contrast, Y76W, with a bulky indole group, was the most stable. These mutants showed higher melting temperatures than WT as a consequence of increased hydrophobic interactions. These results suggest that the tyrosine residue, Y76, is preferred for the cold adaptation of SpGrx4 with a heat‐labile structure despite the rigid cis‐proline loop, due to hydrogen bond formation. An aromatic residue on β3 (cis‐proline plus3) modulates the stability‐flexibility of the cis‐proline loop for temperature adaptation of prokaryotic class II Grx4 members via hydrogen bonds and hydrophobic interactions.

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Effect of active-site aromatic residues Tyr or Phe on activity and stability of glucose 6-phosphate dehydrogenase from psychrophilic Arctic bacterium Sphingomonas sp.

Cited by 9 publications

References 40 publications

Essential roles of buried phenylalanine in the structural stability of thioredoxin from a psychrophilic Arctic bacterium Sphingomonas sp.

Essential roles of buried phenylalanine in the structural stability of thioredoxin from a psychrophilic Arctic bacterium Sphingomonas sp.

Enhancing lactose recognition of a key enzyme in 2′‐fucosyllactose synthesis: α‐1,2‐fucosyltransferase

Tyr76 is essential for the cold adaptation of a class II glutaredoxin 4 with a heat‐labile structure from the Arctic bacteriumSphingomonassp.

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