Site-directed mutagenesis studies on a novel dual domain β-galactosidase/ β-glucosidase open reading frame identified from a dairy run-off metagenome
Date
2021-12
Authors
Journal Title
Journal ISSN
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Publisher
Stellenbosch : Stellenbosch University
Abstract
ENGLISH ABSTRACT: Glycosyl hydrolases (GHs, EC 3.2.1.-) are currently represented by 171 distinct families
(www.CAZY.org) and catalyse the hydrolysis of glycosidic bonds present in various
biological molecules termed glycosides. This generally leads to the release of a
carbohydrate glycone and a carbohydrate or non-carbohydrate aglycon. Glycosidic bonds
can exist between two or more carbohydrate moieties or a carbohydrate moiety and a non-
carbohydrate moiety. Generally, the hydrolysis of the glycosidic bond is catalysed by two
amino acid residues: a general acid/base and a nucleophile.
The β-glucosidases (EC 3.2.1.21) are ubiquitous enzymes that catalyse the hydrolysis of
the β(1-4)-glycosidic bond of glucose-linked carbohydrates to release non-reducing terminal
glucosyl residues, glucosides, or glucooligosaccharides. β-glucosidases are a widely
distributed class of hydrolytic enzymes, existing in every living domain. The GH family 1
represents the largest and most widely studied of the β-glucosidases. Sequence alignment
and site-directed mutagenesis of various GH1 enzymes have revealed two highly conserved
amino acid motifs present in β-glucosidases (TENG and NEP), representing the catalytic
nucleophile and general acid/base catalyst, respectively. Crystallographic analyses of GH1
family β-glucosidases also indicate the presence of a TIM-barrel (β/α8) protein architecture
containing the active site within a deep pocket.
The β-galactosidases (EC 3.2.1.23) are ubiquitous enzymes that catalyse the hydrolysis of
the β(1-4)-linked glycosidic bond of galactose-linked carbohydrates to release non-reducing
terminal galactosyl residues, galactosides or galactooligosaccharides. The β-galactosidase
from Escherichia coli (LacZ), a GH2 enzyme, is currently the best studied β-galactosidase.
Crystallographic and structure mutation studies of the LacZ enzyme shows a tetrameric
protein consisting of four polypeptides that each fold into five distinct domains of which
domain three is a central TIM-barrel of roughly 300 amino acid residues containing the active
site domain. β-galactosidase activity occurs when the catalytic nucleophile (E537) from one
polypeptide overlaps and interacts with the general acid/base catalyst (E461) of another
polypeptide within the respective third domains. This is opposed to the active site within β-
glucosidases, which only requires a single polypeptide chain to achieve activity.
In previous studies, a fosmid-based metagenomic library composed of 40 kB inserts was
derived from the run-off of a dairy farm. Using plate-based functional screening
methodologies, a discrete fosmid clone was identified that demonstrated activities for both
β-galactosidase and β-glucosidase. Subsequently, a single open reading frame (ORF) was iii
identified (BG3L, 1315 bp). When BG3L was isolated and sub-cloned into the bacterial
expression vector pRSetA, it also presented with the dual activity previously observed on
both functional screens and in vitro enzyme assays using crude extracts containing
recombinant protein.
In this study, BG3L was further characterized using a site-directed mutagenesis approach
to determine if we could abolish each activity singly and thereby demonstrating if BG3L
contains two discrete functional domains. Structure-based sequence alignments of the
BG3L protein sequence revealed no obvious structural similarity to GH2 β-galactosidases
but resembled several GH1 enzyme architectures. Molecular models of BG3L were
constructed for comparative structural and docking analysis against lactose, cellobiose and
synthetic 4-nitrophenyl glycosides (artificial substrates). The in silico models were validated
against the available crystal structure of a metagenomic β-glucosidase, PDB: 5XGZ. The
validated models were used to identify amino acid residues potentially involved in enzymatic
activities with Q23, E25, H123 and E399 being identified. A PCR-based site-directed
mutagenesis methodology was then used to abolish either β-glucosidase or β-galactosidase
activity. Plate-based functional screening indicated the abolishment of β-galactosidase
activity (E25S) or both activities (E25Q, H123G). None of the mutations led to an
abolishment of only the β-glucosidase activity. The optimal β-glucosidase activity of the
E25S mutated enzyme (lacking β-galactosidase activity) was observed at pH 6.0 and 45 °C,
which is similar to the WT BG3L (unmutated). The E25S mutation also led to improved
affinity for cellobiose over WT BG3L.
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identified (BG3L, 1315 bp). When BG3L was isolated and sub-cloned into the bacterial
expression vector pRSetA, it also presented with the dual activity previously observed on
both functional screens and in vitro enzyme assays using crude extracts containing
recombinant protein.
In this study, BG3L was further characterized using a site-directed mutagenesis approach
to determine if we could abolish each activity singly and thereby demonstrating if BG3L
contains two discrete functional domains. Structure-based sequence alignments of the
BG3L protein sequence revealed no obvious structural similarity to GH2 β-galactosidases
but resembled several GH1 enzyme architectures. Molecular models of BG3L were
constructed for comparative structural and docking analysis against lactose, cellobiose and
synthetic 4-nitrophenyl glycosides (artificial substrates). The in silico models were validated
against the available crystal structure of a metagenomic β-glucosidase, PDB: 5XGZ. The
validated models were used to identify amino acid residues potentially involved in enzymatic
activities with Q23, E25, H123 and E399 being identified. A PCR-based site-directed
mutagenesis methodology was then used to abolish either β-glucosidase or β-galactosidase
activity. Plate-based functional screening indicated the abolishment of β-galactosidase
activity (E25S) or both activities (E25Q, H123G). None of the mutations led to an
abolishment of only the β-glucosidase activity. The optimal β-glucosidase activity of the
E25S mutated enzyme (lacking β-galactosidase activity) was observed at pH 6.0 and 45 °C,
which is similar to the WT BG3L (unmutated). The E25S mutation also led to improved
affinity for cellobiose over WT BG3L.
To our knowledge, our approach has identified the E25S as the first such mutation within
GH1 enzymes which indicates that the glutamic acid plays a role in modulating β-galactoside
specificity. It has also revealed that the BG3L gene encodes for a unique enzyme which
presents two discrete enzyme activities from a single active domain.
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar
AFRIKAANSE OPSOMMING: Geen opsomming beskikbaar
Description
Thesis (MScAgric)--Stellenbosch University, 2021.
Keywords
Protein modelling, Metagenomics, Beta-glucosidase, Beta-galactosidase, Site-specific mutagenesis, UCTD