Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor Science Reviews - Biology, 2024, 3(1), 16-31
16
Glycosyltransferases: Unraveling Molecular Insights
and Biotechnological Implications
Humara Naz Majeed*,PhD, Sumaira Shaheen, PhD and Muhammad Kashif
Zahoor
1
, PhD
Department of Biochemistry, Government College Women University, Faisalabad, Pakisn
1
Department of Zoology, Government College University Faisalabad, Pakisn
*Dr. Humara Naz Majeed (Corresponding Author)
Department of Biochemistry, Government College Women University, Faisalabad; drhumaranaz@gcwuf.edu.pk
https://orcid.org/0000-0002-9393-1941
1
Dr. Muhammad Kashif Zahoor
Department of Zoology, Government College University, Faisalabad; kashif.zahoor@gcuf.edu.pk
Dr. Sumaira Shaheen
Department of Biochemistry, Government College Women University, Faisalabad; sumerauaf@gmail.com
https://doi.org/10.57098/SciRevs.Biology.3.1.3
Received March 30, 2024. Revised April 17, 2024. Accepted April 18, 2024.
Abstract: Glycosyltransferases (GTs) are present in almost all living organisms; plants, animals and
microorganisms. GTs transfer sugar molecule from nucleotide sugars to a wide range of molecules including
hormones, secondary metabolites, biotic and abiotic chemicals. When glycosyltransferases add a sugar moiety
in any molecule, the hydrophilicity of that molecule changes and thus alter the chemical properties of the
molecule. This phenomenon is vital for appropriate working of living organisms. For the first time, X-ray
structure of bacteriophage T4-glucosyltransferase was reported in 1994. In bacteria, GTs play essential roles in
various biological processes such as cell wall biosynthesis, surface glycosylation and virulence factor
production. The point mutation as well as the domain-swapping has been reported in Bacteria. The sequence
change as well as the whole cells has been engineered in bacteria too. GTs play very important role in survival,
growth, development, metabolism, detoxification, insecticide resistance, chitin formation, chemosensation,
defense and immunity, involved in various signaling pathways, etc. In plants, glycosyltransferase enzymes play
essential role in biosynthesis of cell wall components, secondary metabolites, and signaling molecules. GTs are
involved in the transfer of sugar moieties from activated donor molecules to specific acceptor molecules, leading
to the formation of glycosidic bonds. GTs modify flavonoids, alkaloids and terpenoids, etc. with sugar residues
and alter the solubility, stability, and bioactivity of these compounds and regulate the plant defense mechanism
and interaction with insects, microorganisms and other organisms. GTs have direct impact on plant
homeostasis. Site directed mutagenesis (SDM) in UGTs or GTs cause a change in substrate specificity and
produce increased or total loss in the catalytic activity GTs. This kind of change demonstrated that a change in
substrate specificity could cause better glycosylation and perked up anticancer activity of UGTs. GTs are also
involved in glycosylaton of phytohormones and regulate their metabolism and signaling pathways. GTs are
involved in the activity, stability, and transport of these hormones and influence the plant growth, development,
and responses to various environmental stimuli. Four UGT families encoding 200 genes are reported in humans
which regulate cell signaling, protein folding, immune response, growth and development, detoxification,
metabolism and elimination of drugs, DNA methylation and histone modifications, transcriptional regulation,
post-transcriptional regulation and post-translational regulation, synthesis of human blood group antigens A and
B and recently GTs are also reported as linked with COVID-19-related loss of smell or taste. Various
bioinformatics tools have been developed which would help analyse in silico, the structure of the GTs using any
reference enzyme. The activity and the ordered structures along with various stability assays can be performed
before to conduct in vitro analyses such as mutagenesis. Targeted mutagenesis have been reported through site
Science Reviews - Biology, 2024, 3(1), 16-31 Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor
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directed mutagenesis (SDM) or domain-swapping. Standard protocols of molecular biology i.e. transformation,
protein expression, extraction and purification followed by mass spectrometric analysis has been described. This
molecular technique would direct future endeavours to engineer more glycosyltransferases to augment their
activity with different substrates and provide a basis for more exploration of GTs as an active compound for
potential anti-cancer therapeutics. Additionally, the role of GTs in medicine, food industry, pharmaceutical
industry and agriculture is discussed. More research work is needed for the better understanding of the
biological processes and the mechanisms of glycosyltransferases involved in cancer, tumor, drug metabolism
etc. New era of engineering is awaited to engineer these GT enzymes in vitro to get them boost in industry as
well as to help cure cancer and other diseases as well.
Introduction
Glycosyltransferases (GTs) are present in al-
most all living organisms; plants, animals and mi-
croorganisms (Moremen et al. 2019; Rini et al. 2022;
Andreu et al. 2023). They are involved in glycosyla-
tion reaction and change the hydrophilicity of mol-
ecules in living organisms and helps in detoxifica-
tion and stabilization of natural products (Sa-
kakibara, 2009; Bowles and Lim, 2010; Andreu et al.
2023). The glycosylation, in fact, is responsible for
cell homeostasis and key mechanism catalyzed by
glycosyltransferases to orchestrate bioactivity, me-
tabolism and the position of molecules inside cells
(Offen et al., 2006; Rini et al. 2022). GTs catalyze gly-
cosyl group transfer with inversion or retention of
the anomeric stereochemistry with respect to the
donor sugar (Fig. 1)
Structure and mechanism of
glycosyltransferases
For the first time, X-ray structure of bacterio-
phage T4-glucosyltransferase was reported in 1994
(Vrielink et al., 1994). Later, specific conserved do-
mains were reviewed in glycosyltransferases which
recognize the donor and acceptor molecules. In ad-
dition, the database for homologous sequences was
also studied and the conserved amino acids were
found (Kapitonov and Yu, 1999). The crystal struc-
ture and sequence-based classification of glycosyl-
transferases divided these enzymes into many fam-
ilies which represent the diversity of acceptor mol-
ecules used by these enzymes (Coutinho et al., 2003).
GTs catalyze the biosynthesis of glycosidic bond
during which a nucleoside phosphate sugar is used
as a donor molecule. Two structural folds of GTs
have been reported, i.e. GT-A and GT-B; nucleotide
sugar dependent and lipid phosphosugar-depend-
ent folds, respectively (Breton et al., 2006; Lairson et
al., 2008). New folds were also discovered in bacte-
rial sialytransferase and three dimensional data-
bases were generated to gather the crystal structure
of GTs (Breton et al., 2006). GTs are involved in the
biosynthesis of oligosaccharides, polysaccharides
and glycoconjugates. According to sequence simi-
larity, GTs are divided into 91 families (Kim et al.,
2006). Coordinated action of number of glycosyl-
transferases catalyzes the transfer of sugar residue
from donor molecule to acceptor molecule. Hansen
et al. (2012) revealed that still many protein families
in GTs have not been recognized and reviewed
these unknown glycosyltransferases with their role
in the biosynthesis of cell wall (Hansen et al., 2012).
Three-dimensional model was constructed using
Withania somnifera, which showed that the GTs have
some GT-B type folds (Jadhav et al., 2012).
This enzyme group is very old and the re-
search work related to structure, function and cellu-
lar mechanism of this class of enzymes has not been
extensively performed yet. Moreover, subsequent
kinetic studies have revealed important aspects in
various mechanisms of glycosyltransferases (Breton
et al., 2012). The detailed mechanism of molecular
biology, gene expression and regulation for in vivo
glycosylation is still inadequate for comprehensive
understanding of glycosyltransferases (Kizuka et al.,
2014).
Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor Science Reviews - Biology, 2024, 3(1), 16-31
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Figure 1: Glycosyltransferase and glycosyl transfer mechanism
Glycosyltransferases catalyze glycosyl group transfer with either inversion or retention of the anomeric stereochemis-
try with respect to the donor sugar (Taken from Coutinho et al., 2003)
1. Bacteria and glycosyltransferases
In prokaryotes, glycosylation occur in cyto-
plasm and periplasmic space, whereas in eukary-
otes, it takes place in cytosol, golgi complex and the
endoplasmic reticulum (Liang et al., 2015). In bacte-
ria, GTs play essential roles in various biological
processes such as cell wall biosynthesis, surface gly-
cosylation and virulence factor production (More-
men and Haltiwanger, 2019). These enzymes are in-
volved in the synthesis of polysaccharides, glycoli-
pids, and glycoproteins, which are essential for bac-
terial survival, pathogenicity, and interaction with
the host environment (Yakovlieva & Walvoort, 2019;
Yakovlieva et al., 2021; Rini et al., 2022). GTs catalyze
the transfer of sugar moieties from activated donor
molecules to the specific acceptor molecules, lead-
ing to the formation of glycosidic bonds (Schmid et
al., 2016; Andreu et al., 2023). In Gram-negative bac-
teria, GTs are involved in the assembly of complex
carbohydrate structures, i.e. O-antigen of lipopoly-
saccharides. Similarly, other lipid-linked sugars
serve as donor substrates for bacterial glycosyl-
transferases involved in the assembly of pepti-
doglycan in both Gram-negative and Gram-posi-
tive bacteria (Schmid et al., 2016).
The point mutation as well as the domain-
swapping has been reported in Bacteria. In Helico-
bacter pylori, studies on molecular biology has been
employed to generate twelve fucosyltransferase
chimeras to better understand the regioselectivity of
a-1,3/4-fucosyltransferases. It was found that 347-
353 residues were the key region that regulated a-
1,4 activity (Ma et al., 2003). Further, mutagenesis
analyses of these seven amino acids revealed that
Y350 was responsible for a-1,4 activity. Molecular
studies confirmed the absolute need for an aromatic
residue at this position along with the tyrosine hy-
droxyl group for optimal activity (Ma et al., 2005).
In Pasteurella multocida, engineering through
activity knock-out on hyaluronan synthase was
used to synthesize glycosaminoglycans (Jing &
DeAngelis, 2004). Hyaluronan synthase possesses
two catalytic domains (b-1,3-N-acetylglucosaminyl-
transferase and b-1,4-glucuronosyltransferase ac-
tivities, respectively) and mutation in one domain
caused loss of activity and produced monofunc-
tional GTs. These mutant GTs could be immobilized
and used in alternation for the production glycosa-
minoglycans (Jing & DeAngelis, 2003). In E. coli, en-
gineering of GTs have been reported to synthesize
oligosaccharide moieties of gangliosides GM3, GM2
(Fort et al., 2005). Similarly, mutation at Gln189 in a-
galactosyltransferase from Neisseria meningitides
also resulted in reduced activity of transferase (Lair-
son et al., 2004). Interestingly, beside sequence
change, the whole cells were also engineered by in-
troducing genes for carbohydrate-processing en-
zymes which showed valuable biosynthetic capa-
bilities of GTs to generate large scale production of
oligosaccharides. Herein, the whole cells overex-
press the genes encoding glycosidases, GTs and
sugarnucleotide biosynthetic machinery (Hancock
et al., 2006).
2. Insects and glycosyltransferases
The first ever evidence of glycosyltransferase
activity in insects was obtained from feces of a lo-
cust, Locusta migratoria (Myers and Smith, 1954).
Science Reviews - Biology, 2024, 3(1), 16-31 Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor
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Glycosyltransferases play very important role in
survival, development, metabolism and immunity.
GTs are involved in the biosynthesis of complex car-
bohydrates, glycoproteins, and glycolipids which
are essential for insect growth, reproduction, and
response to environmental challenges (Nagare et al.,
2021). GT-mediated detoxification in most of the in-
sects is a defense strategy against plant allelochem-
icals and xenobiotic compounds. It has been re-
ported that insects evolved a physiological modula-
tion to feed on plants and hence; thereby, glyco-con-
jugation of lipophilic molecules by GTs convert
them into water-soluble products which are then
excreted out of the body (Winde and Wittstock,
2011). GTs are also thought to interact in detoxifica-
tion with other enzymes such as glutathione-S-
transferases (GST), phosphotransferases, sul-
fotransferases, aminotransferases and glycosidases
(Berenbaum and Johnson, 2015). It is worth men-
tioning here that many of these enzyme families are
an outcome of Horizontal Gene Transfer (HGT) be-
tween prokaryotic organisms and arthropod ge-
nome (Wybouw et al., 2016).
GTs are expressed in multiple tissues includ-
ing fat bodies, haemolymph, antennae, midgut, legs,
wings and gonads (Bozzolan et al., 2014). In Bombyx
mori, GTs are expressed in various tissues i.e. testis,
ovary, head, integument, fat body, midgut, haemo-
cyte, malpighian tubules and silk glands (Huang et
al., 2008). Tissue specific expression of GTs have
been reported in the gut in Athetis lepigone moth in-
volved in degrading plant allelochemicals and de-
toxification of insecticide (Zhang et al., 2017). In
some lepidopteran insects i.e. Helicoverpa armigera,
Helicoverpa zea and Helicoverpa assulta; GTs detoxify
capsaicin by glycosylation and help excrete then the
inactivated toxin in the form of capsaicin glucoside
(Ahn et al., 2012). In Myzus persicae nicotianae, RNAi-
mediated silencing revealed that four highly ex-
pressed UGT genes of UGT330A3, UGT344D5,
UGT348A3 and UGT349A3 are required in the de-
toxification of nicotine (Pan et al., 2019). Few con-
served UDP Glycosyltransferases (UGTs) such as
UGT50A1 are expressed throughout the insect body
and also have orthologs in humans (UGT8A1) or
other higher eukaryotes. The conserved and ubiqui-
tous expression of GTs might be involved in glyco-
sylation of cell membrane lipid moieties and play
an important role in the cellular homeostasis (Ahn
et al., 2012).
GTs perform crucial functions in develop-
mental processes of insects such as eye develop-
ment, epithelial development, ommatidia develop-
ment, embryonic development (Chen et al., 2007;
Hagen et al., 2009), tissue differentiation, chitin syn-
thesis for cuticle formation, cuticular tanning and
body pigmentation, UV shielding; homeostasis by
regulating diverse metabolic pathways, neuronal
differentiation, chemosensation, odorant detection,
communication, mate recognition, and defense
against desiccation as well as various external and
internal threats such as predator attacks, physiolog-
ical dysfunctions etc. GTs play significant role in
regulating developmental processes like organo-
genesis, metamorphosis and gametogenesis in in-
sects (Walski et al., 2017). Signaling pathways like
Notch signaling, Hedgehog (Hh) and Decapenta-
plegic (Dpp) in Drosophila are also reported to be
regulated by GTs (Fig. 2) (Ahn et al., 2021; Nagare et
al., 2021). GTs are reported to regulate the mecha-
nism for pesticide cross-resistance (Chen et al., 2019).
GTs are reported constitutively overexpressed in
DDT-resistant Drosophila melanogaster, Carbamate-
resistant Myzus persicae and Neonicotinoid-resistant
Bemisia tabaci (Pedra et al., 2004). Permethrin re-
sistance in Anopheles gambiae and Abamectin re-
sistance in Tetranychus cinnabarinus is mediated via
GTs (Wang et al., 2018; Nagare et al., 2021). In Colo-
rado potato beetle (CPB), Leptinotarsa decemlineata,
UGT2 has been identified as a putative enzyme in-
volved in Imidacloprid resistance (Kaplanoglu et al.,
2017). It has been suggested that the indispensabil-
ity of GTs could make them a potential target in in-
sect pest control strategy via RNAi as a genetic tool
through introduction of dsRNA (Lopez et al., 2019).
Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor Science Reviews - Biology, 2024, 3(1), 16-31
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Figure 2: A summary of the role of GTs in insects
3. Plants and glycosyltransferases
In plants, glycosyltransferase enzymes play
essential role in various biological processes, in-
cluding the biosynthesis of cell wall components,
secondary metabolites, and signaling molecules (He
at al., 2022; Al-Khayri et al., 2023). GTs contribute
significantly in the biosynthesis of cellulose, hemi-
cellulose, and pectins which thereby, impart to
overall structure, function and the integrity of the
cell wall (Guerriero et al., 2018; Al-Khayri et al.,
2023). UDP glycosyltransferases (UGTs) belongs to
family 1 of glycosyltransferases and involved in
glycosylation of wide range of acceptor molecules;
hormones, phenylparanoids, flavonoids, betalains,
coumarins, terpenoids, steroids and glucosinolates
in plants. UGTs have very extensive substrate spec-
ificity for sugar acceptors, so their biochemical anal-
yses helped a lot to understand their functions in
Plants. UGTs are tremendously useful for in vitro
manipulation of UDP sugars. UGTs catalyze the bi-
osynthesis of polysaccharides of cell wall and addi-
tion of N-linked glycans to glycoproteins. In grape
alone, more than 200 different glucosides have been
identified (Sefton et al., 1994). In Arabidopsis thaliana,
120 UGT encoding genes have been identified,
whereas in 244 UGTs are reported in Oryza sativa
(Al-Khayri et al., 2023). The crystal structures of
plant UGTs have also provided the structural basis
for understanding the catalytic mechanism and the
substrate specificity. The crystal-based 3D struc-
tures of four plant UGTs have recently been pub-
lished. Arabidopsis thaliana is the first plant whose
complete genome has been sequenced and served
as a model plant for research work; out of 120 genes,
known functions of many genes are completely
documented (Sakakibara, 2009). GTs perform major
function in plants to modify small molecules and
secondary metabolites i.e. alkaloids flavonoids and
terpenoids with sugar molecules. The glycosylation
thereby regulate the solubility, bioavailability, sta-
bility, and bioactivity of the aforementioned com-
pounds. This process affects the plant defense
mechanism and the interaction with other organism
like insects and animals (Al-Khayri et al., 2023). GTs
involved in glycosylation of plant secondary metab-
olites has a conserved motif of 40 amino acids to-
wards the C-terminus, known as the plant second-
ary product glycosyltransferases (PSPG) box. This
PSPG box in GTs renders them characterisitic func-
tioning in plants (Fig. 3) (Guerriero et al., 2018; Al-
Khayri et al. 2023).
It has been recently reported that GTs contrib-
ute significantly to the mechanism of plant disease
resistance i.e. tobacco mosaic virus (TVM) and Pseu-
domonas syringae inoculation in Nicotiana tabacum
and CaUGT1 in Capsicum annuum. A detailed study
has been conducted by Majeed to elucidate the ac-
tivity of UGTs in Arabidopsis thaliana in order to find
novel functions and the potential role of putative
residues to combat Cancer in future (Majeed et al.,
2015). Subsequently, UGT73B3 and UGT73B5 in Ar-
abidopsis thaliana also revealed the role of GTs for re-
sistance against bacterial infection (Campos et al.,
2019).
In plants, GTs are also involved in the
metabolism and signaling pathways of hormones
by glycosylating phytohormones such as auxins,
cytokinins, and gibberellins; and can modulate the
activity, stability, and transport of these hormones
and influence the plant growth, development, and
Science Reviews - Biology, 2024, 3(1), 16-31 Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor
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responses to various environmental stimuli.
Various in vitro studies have facilitated more
knowledge about the multigene family of
glycosyltransferases (Bowles and Lim, 2010; Majeed
et al., 2015). The blended knowledge of
biochemistry, proteomics, genomics, molecular
biology and computer analysis would provide
splendid advancement in plant
glycosyltransferases (Majeed et al., 2015; He at al.,
2022; Al-Khayri et al., 2023).
Figure 3: A summary of the role of GTs in Plants
4. Humans and glycosyltransferases
UDP-glycosyltransferases (UGTs) are phase II
metaboloism enzymes of xenobiotics and use UDP-
glucuronic acid as donar sugar in vertebrates (Dong
et al., 2012; Buchheit et al., 2011). In humans, there
are four UGT families
(UGT1, UGT2, UGT3 and UGT8) and each member
of these families is unique in substrate selection (Hu
et al., 2022). More than 200 glycosyltransferases are
reported which regulate the enzymatic addition of
various carbohydrate molecules in human cell
(Shimma et al., 2006; Narimatsu et al., 2019). Out of
22 UGTs reported in humans, 19 UGTs have very
distinct substrate specificity. For instance, UDP glu-
curonic acid is mostly accepted donor sugar for hu-
man UGTs (Hu et al., 2022).
GTs in humans are involved in numerous bi-
ological processes such as cell signaling, protein
folding, immune response and the development
(Schmid et al., 2016; Rini et al., 2022). Aberrant pro-
tein glycosylation leads to cancer, autoimmune dis-
orders and/ or congenital disorders of glycosyla-
tion (Meech et al., 2015). GTs biosynthesize glyco-
proteins, glycolipids and glycosphingolipids which
help regulate cell signaling, cell-cell recognition and
communication, and adhesion (Ryckman et al., 2020;
Jaroentomeechai et al., 2022). GTs are also reported
to biosynthesize mucins which lubricate and pro-
tect mucosal surfaces (Grondin et al., 2020). UGTs
regulate the metabolism and elimination of human
hydrophilic drugs and chemical substances. UGTs
conjugate lipophilic compounds to sugars i.e. glu-
curonide, galactose, glycosyl, or galacto; with sub-
strates such as cancer-causing substances, medica-
tions, corticosteroids, triglycerides, fatty acid oxida-
tion and bile salts etc. (Meech et al., 2015). Through
glucuronidation process, UGTs weaken the biolog-
ical activity of these drugs or chemical substances
and increases their water solubility; hence, driving
them to be eliminated in bile, urine and feces (Liu et
al., 2023). Human UGTs are expressed in a wide
range of organs and tissues. Mostly prominent in
the liver, kidney and intestine; hence, reflecting
their role in detoxification (Mazerska et al. 2016; Liu
et al., 2023). UGTs are also involved in epige-
netic modifications such as DNA methylation and
histone modifications, transcriptional regulation,
post-transcriptional regulation (miRNA), and post-
translational regulation i.e. structural and func-
tional modifications, and protein-protein interac-
tions (Yasar et al. 2013; Hu et al., 2014; Hu et al., 2019).
GTs are responsible for the synthesis of hu-
man blood group antigens A and B (a-1,3-N-acetyl-
galactosaminyltransferase and a-1,3-galactosyl-
transferase, respectively); which are shown to differ
by only four amino acids (R/G176, G/S235,
L/M266 and G/A268). Glycosyltransferase adds an
immunodominant carbohydrate to the H antigen.
Blood group A (A phenotype) results from the 3-α-
N-acetylgalactosaminyltransferase which adds
Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor Science Reviews - Biology, 2024, 3(1), 16-31
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GalNAc to the H antigen; whereas, Blood group B
(B phenotype) is results from the 3-α-galactosami-
nyltransferase that adds galactose (Gal) to the H an-
tigen. Blood group AB (AB phenotype) has both the
enzyme activities. Blood group O (O phenotype, re-
cessive) has no functional enzyme due to a prema-
ture stop codon in the gene (Delaney, 2013). GTs has
been recently reported to be linked with COVID-19-
related loss of smell or taste (Shelton et al., 2022).
Understanding the role of GTs needs extensive mo-
lecular biology and biotechnological work in order
to elucidating various processes and disease mech-
anisms.
In Silico modeling of glycosyltransferases through
Bioinformatics tools
Valuable information on enzyme-substrate
interactions is available, which is obtained from
crystal structures of GTs in complex with different
acceptor- or donor-substrates or analogs (McArthur
and Chen, 2016). However, bioinformatics tools for
accurate prediction of sequence features important
for defining the substrate specificity of a particular
GTs have been devised (Majeed, 2014; Kuhlman
and Bradley, 2019). In bacteria, a program was de-
veloped using available biochemical and crystal
structure data to predict acceptors for GTs that gly-
cosylate antibiotics (Kamra et al., 2005). Higher
amino acid sequence identity between the query se-
quence and the template; as well as substantial
amounts of biochemical data are essential elements
to achieve reliable prediction (Jabeen et al., 2019;
Kuhlman and Bradley, 2019; Jumper et al., 2021; Sas-
idharan & Saudagar, 2022). The 3D glycosyltrans-
ferase database holds crystal structures of 53 differ-
ent GTs (http://www.cermav.cnrs.fr/glyco3d/).
Furthermore, few other crystal structures can be
found at the RCSB protein data bank
(http://www.rcsb.org/pdb).
In silico prediction modeling approaches can
be categorized as template-based modeling (TBM)
or template-free modeling (TFM). Normally, best
templates are searched out from the Protein Data
Bank (PDB) library using TBM method (Pearce et al.,
2021). Thus, before to perform targeted mutation or
site directed mutagenesis (SDM); these prediction
models help predict the potential mutation site
(Majeed, 2014). The template search with Blast per-
formed against SWISS-MODEL template library
(SMTL) (Bienert et al., 2017). The target sequence
can be searched with BLAST against the primary
amino acid sequence contained in the SMTL
(Altschul et al., 1997; Majeed, 2014). The predicted
highest quality template has been identified and,
subsequently selected for model building using
UCSF Chimera (Meng et al., 2006). Further, the su-
perimposition of UGT with reference enzyme i.e.
VvGT1 was performed to reveal the target mutation
site (Majeed, 2014). Mostly, the disordered proba-
bility for all the Amino Acids has been analyzed
through PrDOS (http://prdos.hgc.jp/cgi-
bin/top.cgi). Ramachandran plot, introduced by an
Indian Physicist G. N. Ramachandran is reported to
visualize the back bone of polypeptide chain, to cal-
culate the phi and psi angles and also for structural
validation (Ramachandran and Sasisekharan 1968;
Singh, 2012). The mutated or target amino acid falls
in allowed region; meaning thereby an indication of
stability in protein structure which can be worked
out for further molecular biology and biotechnolog-
ical engineering (Singh, 2012).
Molecular Biology and Engineering of Glycosyl-
transferases
Biochemical characterization of the wide
range substrate specificity of the GTs is a major task
to gain a thorough understanding of the specificity
of individual glycosyltransferase enzyme. When
the substrate profile for a GT has been determined,
mutational analyses may provide valuable infor-
mation regarding the role of single amino acid resi-
dues with respect to sugar acceptor and donor spec-
ificity and catalytic efficiency (Ramakrishnan et al.,
2008). Glycosyltransferases have specific domains
which regulates the process of glycosylation in re-
actant molecules. The addition of a sugar molecule
from donor sugar to acceptor molecule by glycosyl-
transferases during glycosylation reaction ulti-
mately changes the hydrophilicity and bioactivity
of acceptor molecules (Offen et al., 2006). Hence,
GTs engineering would help to understand the do-
main specificity for their functionality in a given bi-
ological process (He et al., 2022; Rini et al., 2022; An-
dreu et al., 2023; Liu et al., 2023).
The structure-based UGT engineering can al-
ter substrate specificity; compromise or enhance
catalytic efficiency; and confer reversibility to the
glycosylation reaction. It not only changes the sub-
strate specificity but also increases or decreases
their catalytic activity and may lead to totally inac-
tive enzyme or the enzyme with enhanced activity
(Wang, 2009).
Enzymatic engineering of original enzymes
either by SDM or domain swapping is an