Crystal structure of a novel two domain <scp>GH</scp>78 family α‐rhamnosidase from <scp><i>K</i></scp><i>lebsiella oxytoca</i> with rhamnose bound

O’Neill, Ellis C.; Stevenson, Clare E. M.; Paterson, Michael; Rejzek, Martin; Chauvin, Anne-Laure; Lawson, David M.; Field, Robert A.

doi:10.1002/prot.24807

Cited by 36 publications

(32 citation statements)

References 32 publications

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“…Based on the recently resolved 3D structure of the SaRha78A (PBD: 3W5N), four aromatic residues (Trp 640 , Trp 695 , Tyr 744 , and Trp 747 ) are involved in a hydrophobic pocket formation in the catalytic module, and substrate rhamnose has direct and solvent-mediated hydrogen bonds with nine residues (Glu 636 , Arg 543 , Asp 630 , His 916 , Arg 634 , Asp 643 , Trp 695 , Trp 747 , and Glu 895 ) (Fujimoto et al 2013). The similar structure is also observed in BsRhaB and KoRha (Cui et al 2007;O'Neill et al 2015). Crystal structures of SaRha78A and KoRha enzyme-substrate complexes enabled the precise localization of the active site.…”

supporting

confidence: 63%

“…The 3D modeling result of RhaL1 also demonstrates an (α/α) 6 -barrel structure (Fig. 2a) which displayed significant structural similarity to the catalytic module of BsRhaB, SaRha78A, and KoRha (Cui et al 2007;Fujimoto et al 2013;O'Neill et al 2015), and the substrate rhamnose was located in the deep cleft of the (α/α) 6 -barrel (Fig. 2xa).…”

Section: Construction Of Rhal1 Mutantsmentioning

confidence: 88%

“…So far, four crystal structures of GH78 α-L-rhamnosidases have been solved and these solved structures provide valuable information on structure simulation and mutation strategy design of enzymes within the same family. From the solved crystal structures, rhamnosidases from GH78 possess a typical catalytic (α/α) 6 -barrel domain (Cui et al 2007;Fujimoto et al 2013;O'Neill et al 2015) containing some residues, such as Asp 567 , Glu 572 , Trp 576 , and Asp 579 in BsRhaB, and are completely conserved (Cui et al 2007). The 3D modeling result of RhaL1 also demonstrates an (α/α) 6 -barrel structure (Fig.…”

Section: Construction Of Rhal1 Mutantsmentioning

confidence: 98%

“…Crystal structures of SaRha78A and KoRha enzyme-substrate complexes enabled the precise localization of the active site. With the accumulation of structural information, the stacking residues and the residues forming hydrogen bonds with rhamnose in the active site are supposed to be critical for the enzymatic activity (Fujimoto et al 2013;O'Neill et al 2015). However, few attempts have been made to probe the influence of these residues on rhamnosidase activity.…”

mentioning

confidence: 99%

“…To date, four crystal structures of α-L-rhamnosidases have been solved-putative α-L-rhamnosidase BT1001 from Bacteroides thetaiotaomicron VPI-5482 (Bonanno et al 2005), α-L-rhamnosidase B (BsRhaB) from Bacillus sp. GL1 (Cui et al 2007), α-L-rhamnosidase (SaRha78A) from Streptomyces avermitilis (Fujimoto et al 2013), and putative α-L-rhamnosidase (KoRha) from Klebsiella oxytoca (O'Neill et al 2015)-and all these enzymes belong to GH78. The family GH78 rhamnosidases catalyze the hydrolysis of α-Lrhamnosyl linkages through an acid-base-assisted single displacement (inverting) mechanism (Yadav et al 2010).…”

mentioning

confidence: 99%

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Site-directed mutagenesis of α-l-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design

Liu

Yin³

et al. 2016

Appl Microbiol Biotechnol

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The α-L-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-L-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp, Asp, Asp, Glu, Arg, His, and Trp) and may form the hydrophobic pocket in stabilizing donor (Trp, Tyr, Tyr, and Trp) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7 %) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp. Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.

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supporting

confidence: 63%

Section: Construction Of Rhal1 Mutantsmentioning

confidence: 88%

Section: Construction Of Rhal1 Mutantsmentioning

confidence: 98%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Site-directed mutagenesis of α-l-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design

Liu

Yin³

et al. 2016

Appl Microbiol Biotechnol

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An effective computational‐screening strategy for simultaneously improving both catalytic activity and thermostability of α‐l‐rhamnosidase

Gong

et al. 2021

Biotech & Bioengineering

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Catalytic efficiency and thermostability are the two most important characteristics of enzymes. However, it is always tough to improve both catalytic efficiency and thermostability of enzymes simultaneously. In the present study, a computational strategy with double‐screening steps was proposed to simultaneously improve both catalysis efficiency and thermostability of enzymes; and a fungal α‐l‐rhamnosidase was used to validate the strategy. As the result, by molecular docking and sequence alignment analysis within the binding pocket, seven mutant candidates were predicted with better catalytic efficiency. By energy variety analysis, A355N, S356Y, and D525N among the seven mutant candidates were predicted with better thermostability. The expression and characterization results showed the mutant D525N had significant improvements in both enzyme activity and thermostability. Molecular dynamics simulations indicated that the mutations located within the 5 Å range of the catalytic domain, which could improve root mean squared deviation, electrostatic, Van der Waal interaction, and polar salvation values, and formed water bridge between the substrate and the enzyme. The study indicated that the computational strategy based on the binding energy, conservation degree and mutation energy analyses was effective to develop enzymes with better catalysis and thermostability, providing practical approach for developing industrial enzymes.

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Aspergillus oryzae α‐l‐rhamnosidase: Crystal structure and insight into the substrate specificity

Makabe,

Ishida,

Kanezaki

et al. 2023

Proteins

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The subsequent biochemical and structural investigations of the purified recombinant α‐l‐rhamnosidase from Aspergillus oryzae expressed in Pichia pastoris, designated as rAoRhaA, were performed. The specific activity of the rAoRhaA wild‐type was higher toward hesperidin and narirutin, where the l‐rhamnose residue was α‐1,6‐linked to β‐d‐glucoside, than toward neohesperidin and naringin with an α‐1,2‐linkage to β‐d‐glucoside. However, no activity was detected toward quercitrin, myricitrin, and epimedin C. rAoRhaA kinetic analysis indicated that Km values for neohesperidin, naringin, and rutin were lower compared to those for hesperidin and narirutin. kcat values for hesperidin and narirutin were higher than those for neohesperidin, naringin, and rutin. High catalytic efficiency (kcat/Km) toward hesperidin and narirutin was a result of a considerably high kcat value, while Km values for hesperidin and narirutin were higher than those for naringin, neohesperidin, and rutin. The crystal structure of rAoRhaA revealed that the catalytic domain was represented by an (α/α)6‐barrel with the active site located in a deep cleft and two β‐sheet domains were also present in the N‐ and C‐terminal sites of the catalytic domain. Additionally, five asparagine‐attached N‐acetylglucosamine molecules were observed. The catalytic residues of AoRhaA were suggested to be Asp254 and Glu524, and their catalytic roles were confirmed by mutational studies of D254N and E524Q variants, which lost their activity completely. Notably, three aspartic acids (Asp117, Asp249, and Asp261) located at the catalytic pocket were replaced with asparagine. D117N variant showed reduced activity. D249N and D261N variants activities drastically decreased.

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Crystal structure of a novel two domain GH78 family α‐rhamnosidase from Klebsiella oxytoca with rhamnose bound

Cited by 36 publications

References 32 publications

Site-directed mutagenesis of α-l-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design

Site-directed mutagenesis of α-l-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design

An effective computational‐screening strategy for simultaneously improving both catalytic activity and thermostability of α‐l‐rhamnosidase

Aspergillus oryzae α‐l‐rhamnosidase: Crystal structure and insight into the substrate specificity

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