Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor Science Reviews - Biology, 2024, 3(1), 16-31

Glycosyltransferases: Unraveling Molecular Insights

and Biotechnological Implications

Humara Naz Majeed*,PhD, Sumaira Shaheen, PhD and Muhammad Kashif

Zahoor

, PhD

Department of Biochemistry, Government College Women University, Faisalabad, Pakistán

Department of Zoology, Government College University Faisalabad, Pakistán

*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

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

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

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

sugar–nucleotide 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

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

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

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

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

Science Reviews - Biology, 2024, 3(1), 16-31 Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor

authoritative tool for determination of actual amino

acids present in the catalytic site and it is also help-

ful in modifying enzyme action (Katoh et al., 2004;

Majeed et al., 2015). Site directed mutagenesis in

highly conserved Serine 134 to Leucine of UGT74B1

of Arabidopsis showed very mild morphological and

metabolic changes. Nevertheless, the mutated Ser-

ine showed altered affinity for substrate, UDP-glu-

cose (Kopycki et al., 2013). A mutational study of the

A. cordata UGT78A2 identified a single amino acid

residue responsible for determining UDP-galactose

versus dual UDP-galactose and UDP-glucose spec-

ificity (Kubo et al., 2004). Sulfolobus solfataricus β-

glycosidase has been modified by alteration of two

residues which are involved in substrate recogni-

tion and this modification helped to accept different

substrates in transglycosylation reactions (Hancock

et al., 2006). In case of UGT85B1 of Sorgham bicolor,

attempts to convert glucosyltransferases to glucu-

ronosyltransferases by mutating the corresponding

residues was not successful which showed that

UGTs may require multiple amino acids to recog-

nize sugars although single residues may play a

decisive role (Osmani et al., 2008). Subsequently, the

whole N-terminal domain of UGT74F2 was fused to

the C-terminal domain of UGT74F1 and the chimera

displays UGT74F2-like kinetic parameters and regi-

ospecificity toward the quercetin acceptor. This do-

main swapping approach identified an amino acid

distal to the active site which is important for deter-

mining the regiospecificity of UGT74F1 (Cartwright

et al., 2008).

GT engineering causes the change of color of

flowers; elevate the production of bioactive glyco-

sides and higher tolerance in plants against any

stress (Bowles et al., 2005 and 2006). Enzymatic en-

gineered curumin glycosyltransferase (CaUGT2) by

domain swapping and SDM found to improve the

catalytic activity of CaUGT2. The CaUGT2 mutants

with functionally important Cys377 site also

showed similar Km value as the wild type CaUGT2

(Masada et al., 2010). The engineered AtUGT78D2

and AtUGT78D3 from Arabidopsis thaliana through

domain swapping made these enzymes catalyti-

cally more efficient with extended sugar selectivity

(Kim et al., 2013).

Figure 4: Schematic diagram of various steps in Modeling, Molecular Biology and Engineering of Glycosyltrans-

ferases

Upper panel: In silico modeling and stability assay for glycosyltransferases

Lower Left panel: Transformation for glycosyltransferases by heat shock method

Lower Middle panel: Protein Expression, Extraction and Purification for glycosyltransferases

Lower right panel: Mutagenesis protocol in glycosyltransferases

The development of new UGTs through enzy-

matic engineering also alters the regiospecificity of

UGTs and pattern of glycosylation. In short, by

playing with UGTs in this way improves not only

the efficiency of the enzyme but also changes the

whole cell bioactivity (Lim et al., 2005; Lim, 2005). In

pigs, a point mutation in α-1,3-galactosyltransferase

leads to failure in the synthesis of Gala-1,3-Gal

epitope; which is responsible of hyperacute rejec-

tion in pig-to primate xeno-grafts (Phelps et al.,

2003). Similarly, chimeric GTs were created using

mouse-derived retroviral vectors and this Gala-1,3-

Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor Science Reviews - Biology, 2024, 3(1), 16-31

Gal epitope was reduced as it was not recognized

by the human immune system (Hansen et al., 2005)..

Importance and Applications of Glycosyltransfer-

ases

Glycosyltransferases and cancer treatment

Cancer is one of the major causes of death

these days. The rise in population has laid more

global burden of cancer which is continuously in-

creasing due to the adoption of cancer-associated

lifestyle such as smoking, decreased physical activ-

ity and choice of diets. As GTs are involved in the

secondary metabolism and remove toxins, drugs

and dangerous chemicals including carcinogens

from the body through glycosylation; hence, the ex-

acerbated glycosylation contributes towards inci-

dence of cancer (Gupta et al., 2020; Hu et al., 2022;

Pucci et al., 2022; Liu et al., 2023). There is an inhibi-

tory metabolic effect of N-acetylglucosamine (Glc-

NAc) on human prostate cancer and this was an il-

lustration towards drug development by targeting

GTs (Nishimura et al., 2012). The Cancer Genome

Atlas (TCGA) data revealed that glycosylation

changes thereby the expression pattern of glycosyl-

transferases are linked with Cancer (Pucci et al.,

2022). Conventional therapies i.e., hormonal ther-

apy, surgery, immunotherapy and anti-angiogene-

sis therapy are deficit in actual effectiveness (Hu

and Fu, 2012). The most promising feature reported

for cancer is anomalous glycosylation which

changed the expression of glycosyltransferases

(Meany and Chan, 2011). Hence, due to crucial role

of glycosyltransferases in biological system, GTs

have drawn extraordinary attention of researchers

to develop new drugs by using these enzymes

(Gupta et al., 2020; Pucci et al., 2022). The develop-

ment of glycoproteomic technology is indispensible

because of its higher sensitivity and specificity.

More importantly, this technology has been consid-

ered as reproducible without any reservation (He et

al., 2024). These metabolic inhibitors of glycosyl-

transferases have probability to be focused in drug

discovery. Thus, advanced research in glycomics

would unveil the pathological role of glycosyltrans-

ferases as potential biomarkers and novel targets

would lead to therapeutic use of GTs (Gupta et al.,

2020; Pucci et al., 2022; He et al., 2024).

Glycosyltransferases and Industrial applications

Glycosylation process serves as an important

utility in food industry and the pharmaceutical in-

dustry as well (He et al., 2022). Glycosylation helps

in stabilization of various natural compounds, i.e.

Vitamin C (L-ascorbic acid), an essential nutrient for

humans and certain other animal species. It has

many biological functions such as collagen synthe-

sis, antioxidation, and intestinal absorption of iron.

It is widely used in medicine as an antioxidant and

also as an additive in food industry (Kumar et al.

2016). Because GTs precisely modify the protein

structures which is an hallmark for stability and the

biological activity; and hence, are also reported hav-

ing been used in the cosmetics, skin care and phar-

maceutical industry (Schwab et al., 2015). Interest-

ingly, GTs are also involved in deglycosylation,

which makes these enzymes helpful in biosynthesis

of many activated sugars (Rini et al., 2022; Andreu

et al., 2023). GTs are reported with their use with

certain food items and beverages to improve the

taste as sweeteners and flavor (He et al., 2022; An-

dreu et al., 2023). An environmental friendly and

more advantageous use of GTs is also reported for

biofuel production (Greene et al., 2015; Schwab et al.,

2015). In nutshell, glycosyltransferase enzymes are

versatile enzymes which confer significant contri-

bution across various industries.

Glycosyltransferases and Agriculture

Glycosyltransferase has been reported as

ubiquitous in plants and used to enhance the plant

physiology, adaptation and to improve the stress

tolerance in plants. Moreover, GTs also reported to

help improve the crop protection (Gharabli et al.,

2023). Numerous bioactive compounds are being

produced using glycosylation and biotechnological

engineering (He et al., 2022; Andreu et al., 2023).

Thanks to the insect cell lines, recombinant glyco-

proteins in insect cell expression systems have been

produced (Geisler et al., 2015). To devise a best con-

trol strategy for integrated control of insects, under-

standing of the molecular biology of GT is essential.

Thus, the genetic control strategy for insect pest

management via RNAi has been employed (Lopez

et al., 2019). Additionally, the insecticide resistance

reported in various insects have been managed

which could serve as a tool to be exploited further

in other agricultural crops (Kaplanoglu et al., 2017;

Wang et al., 2018; Chen et al., 2019; Nagare et al.,

2021).

Science Reviews - Biology, 2024, 3(1), 16-31 Humara Naz Majeed, Sumaira Shaheen, Muhammad Kashif Zahoor

Conclusions & Future Perspectives

Glycosyltransferase engineering has been at-

tempted in both prokaryotic and eukaryotic organ-

isms. GTs are involved in a number of biological

processes and connected to various diseases that

shows the functional diversity and the versatility of

GTs. In prokaryotes, GTs regulate cell wall biosyn-

thesis, surface glycosylation and virulence, survival,

pathogenicity, and interaction with the host envi-

ronment. Whereas, in multicellular organism, i.e.

insect; GTs being ubiquitously expressed in multi-

ple tissues; paly significant role in survival, growth

and development, metabolism and immunity, re-

production, detoxification, insecticide resistance,

cuticle formation, homeostasis, chemosensation,

odorant detection, communication, mate recogni-

tion, metamorphosis and gametogenesis, signaling

pathways, etc. In plants,

GTs are component of cell wall and help regulate

metabolisms through sysnthesis of secondary me-

tabolites and signaling molecules. GTs are involved

in glycosylation of wide range of acceptor mole-

cules and show specificity for sugar acceptors. GTs

contribute towards plant disease resistance, plant

growth, development, and responses to various en-

vironmental stimuli. More than 200 glycosyltrans-

ferases are reported in humans which regulate the

enzymatic addition of various carbohydrate mole-

cules and regulate numerous biological processes

such as cell signaling, protein folding, immune re-

sponse and the development, cell signaling, detoxi-

fication, epigenetic modifications such as DNA

methylation and histone modifications, transcrip-

tional regulation, post-transcriptional regulation,

synthesis of human blood group antigens, etc and

recently known to be liked with COVID-19-related

loss of smell or taste. The use of bioinformatics tools

for modeling, and in silico stability assays are sug-

gested as best approach before to perform in vivo GT

analyses and engineering.

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 en-

gineering 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.

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