Dataset Information

Mutational Analysis of the Role of the Glucansucrase Gtf180-?N Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate.

ABSTRACT: Glucansucrase Gtf180-?N from Lactobacillus reuteri uses lactose as acceptor substrate to synthesize five glucosylated lactose molecules (F1-F5) with a degree of polymerization (DP) of 3-4 (GL34) and with (?1?2)/(?1?3)/(?1?4) glycosidic linkages. Q1140/W1065/N1029 mutations significantly changed the GL34 product ratios. Q1140 mutations clearly decreased F3 3'-glc-lac with an (?1?3) linkage and increased F4 4',2-glc-lac with (?1?4)/(?1?2) linkages. Formation of F2 2-glc-lac with an (?1?2) linkage and F4 was negatively affected in most W1065 and N1029 mutants, respectively. Mutant N1029G synthesized four new products with additional (?1?3)-linked glucosyl moieties (2xDP4 and 2xDP5). Sucrose/lactose strongly reduced Gtf180-?N hydrolytic activity and increased transferase activity of Gtf180-?N and mutant N1029G, in comparison to activity with sucrose alone. N1029/W1065/Q1140 thus are key determinants of Gtf180-?N linkage and product specificity in the acceptor reaction with lactose. Mutagenesis of key residues in Gtf180-?N may allow synthesis of tailor-made mixtures of novel lactose-derived oligosaccharides with potential applications as prebiotic compounds in food/feed and in pharmacy/medicine.

SUBMITTER: Pham H

PROVIDER: S-EPMC6328278 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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Publications

Mutational Analysis of the Role of the Glucansucrase Gtf180-ΔN Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate.

Pham Hien H Pijning Tjaard T Dijkhuizen Lubbert L van Leeuwen Sander S SS

Journal of agricultural and food chemistry 20181115 47

Glucansucrase Gtf180-ΔN from Lactobacillus reuteri uses lactose as acceptor substrate to synthesize five glucosylated lactose molecules (F1-F5) with a degree of polymerization (DP) of 3-4 (GL34) and with (α1→2)/(α1→3)/(α1→4) glycosidic linkages. Q1140/W1065/N1029 mutations significantly changed the GL34 product ratios. Q1140 mutations clearly decreased F3 3'-glc-lac with an (α1→3) linkage and increased F4 4',2-glc-lac with (α1→4)/(α1→2) linkages. Formation of F2 2-glc-lac with an (α1→2) linkage ...[more]

PMID: 30396274

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Project description:Cytochrome P450s (CYPs) are a family of enzymes responsible for metabolizing nearly 80% of small molecule drugs. Variants in CYPs can substantially alter drug metabolism, which may result in improper dosing and severe adverse drug reactions. CYPs have low sequence conservation, making it difficult to anticipate whether variant effects measured in one CYP may extend to others based on sequence alone. Even closely related CYPs, like CYP2C9 and its closest homolog CYP2C19, have distinct phenotypic properties despite sharing 92% of their sequences. Thus, we used Variant Abundance by Massively Parallel sequencing (VAMP-seq) to measure the protein abundance of 7,660 missense variants in CYP2C19 expressed in cultured human cells. Our results confirmed positions and structural features critical for CYP function and revealed how variants at positions conserved across all eukaryotic CYPs influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core, and that substitutions in some regions reduced abundance in CYP2C19 but not CYP2C9. We also measured the abundance of all single and some multiple WT amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 (SRS4) reduced abundance in CYP2C19. When nearby amino acids were exchanged in double and triple mutants, we found distinct interactions between the sites in CYP2C19 and CYP2C9, revealing a region that is partially responsible for the difference in thermodynamic stability between the two homologs. Since these positions are also important for determining substrate specificity, there may be an evolutionary tradeoff between stability and altered enzymatic function. Finally, we used our data to analyze 368 previously unannotated human variants, finding that 43% had decreased abundance. Thus, by comparing variant effects between two closely related, important human genes, we have uncovered regions underlying their functional differences and paved the way for a more complete understanding of one of the most versatile families of enzymes.

Dataset Information

Mutational Analysis of the Role of the Glucansucrase Gtf180-?N Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate.

Publications

Mutational Analysis of the Role of the Glucansucrase Gtf180-ΔN Active Site Residues in Product and Linkage Specificity with Lactose as Acceptor Substrate.

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