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Structure-guided engineering of the substrate specificity of a fungal ?-glucuronidase toward triterpenoid saponins.

ABSTRACT: Glycoside hydrolases (GHs) have attracted special attention in research aimed at modifying natural products by partial removal of sugar moieties to manipulate their solubility and efficacy. However, these modifications are challenging to control because the low substrate specificity of most GHs often generates undesired by-products. We previously identified a GH2-type fungal ?-glucuronidase from Aspergillus oryzae (PGUS) exhibiting promiscuous substrate specificity in hydrolysis of triterpenoid saponins. Here, we present the PGUS structure, representing the first structure of a fungal ?-glucuronidase, and that of an inactive PGUS mutant in complex with the native substrate glycyrrhetic acid 3-O-mono-?-glucuronide (GAMG). PGUS displayed a homotetramer structure with each monomer comprising three distinct domains: a sugar-binding, an immunoglobulin-like ?-sandwich, and a TIM barrel domain. Two catalytic residues, Glu414 and Glu505, acted as acid/base and nucleophile, respectively. Structural and mutational analyses indicated that the GAMG glycan moiety is recognized by polar interactions with nine residues (Asp162, His332, Asp414, Tyr469, Tyr473, Asp505, Arg563, Asn567, and Lys569) and that the aglycone moiety is recognized by aromatic stacking and by a ? interaction with the four aromatic residues Tyr469, Phe470, Trp472, and Tyr473 Finally, structure-guided mutagenesis to precisely manipulate PGUS substrate specificity in the biotransformation of glycyrrhizin into GAMG revealed that two amino acids, Ala365 and Arg563, are critical for substrate specificity. Moreover, we obtained several mutants with dramatically improved GAMG yield (>95%). Structural analysis suggested that modulating the interaction of ?-glucuronidase simultaneously toward glycan and aglycone moieties is critical for tuning its substrate specificity toward triterpenoid saponins.

SUBMITTER: Lv B

PROVIDER: S-EPMC5767852 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature

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Structure-guided engineering of the substrate specificity of a fungal β-glucuronidase toward triterpenoid saponins.

Lv Bo B Sun Hanli H Huang Shen S Feng Xudong X Jiang Tao T Li Chun C

The Journal of biological chemistry 20171116 2

Glycoside hydrolases (GHs) have attracted special attention in research aimed at modifying natural products by partial removal of sugar moieties to manipulate their solubility and efficacy. However, these modifications are challenging to control because the low substrate specificity of most GHs often generates undesired by-products. We previously identified a GH2-type fungal β-glucuronidase from <i>Aspergillus oryzae</i> (<i>P</i>GUS) exhibiting promiscuous substrate specificity in hydrolysis of ...[more]

PMID: 29146597

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Project description:BackgroundAldehyde-deformylating oxygenase (ADO) is an important enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, ADO exhibits quite low chain-length specificity with respect to the substrates ranging from C4 to C18 aldehydes, which is not suitable for producing fuels with different properties or different chain lengths.ResultsBased on the crystal structures of cADOs (cyanobacterial ADO) with substrate analogs bound, some amino acids affecting the substrate specificity of cADO were identified, including the amino acids close to the aldehyde group and the hydrophobic tail of the substrate and those along the substrate channel. Using site-directed mutagenesis, selected amino acids were replaced with bulky ones introducing steric hindrance to the binding pocket via large functional groups. All mutants were overexpressed, purified and kinetically characterized. All mutants, except F87Y, displayed dramatically reduced activity towards C14,16,18 aldehydes. Notably, the substrate preferences of some mutants towards different chain-length substrates were enhanced: I24Y for n-heptanal, I27F for n-decanal and n-dodecanal, V28F for n-dodecanal, F87Y for n-decanal, C70F for n-hexanal, A118F for n-butanal, A121F for C4,6,7 aldehydes, V184F for n-dodecanal and n-decanal, M193Y for C6-10 aldehydes and L198F for C7-10 aldehydes. The impact of the engineered cADO mutants on the change of the hydrocarbon profile was demonstrated by co-expressing acyl-ACP thioesterase BTE, fadD and V184F in E. coli, showing that n-undecane was the main fatty alkane.ConclusionsSome amino acids, which can control the chain-length selectivity of substrates of cADO, were identified. The substrate specificities of cADO were successfully changed through structure-guided protein engineering, and some mutants displayed different chain-length preference. The in vivo experiments of V184F in genetically engineered E. coli proved the importance of engineered cADOs on the distribution of the fatty alkane profile. The results would be helpful for the production of fatty alk(a/e)nes in cyanobacteria with different properties.

Dataset Information

Structure-guided engineering of the substrate specificity of a fungal ?-glucuronidase toward triterpenoid saponins.

Publications

Structure-guided engineering of the substrate specificity of a fungal β-glucuronidase toward triterpenoid saponins.

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