Humara Naz Majeed, Aftab Ahmad Science Reviews - Biology, 2024, 3(3), 1-15
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Zeymer and Hilvert, 2018; Fu et al., 2024; Majeed et
al., 2024). The selection and identification of the spe-
cific site for SDM is critical to the process. Various
bioinformatics tools and software not only help in
identifying the precise mutagenesis site but also in
reproducing 3D structures before and after muta-
genesis (Majeed et al., 2024a; Steiner et al., 2012).
Databases like Swiss-model or NCBI can be used to
generate amino acid sequences for drawing 3D
structures. Through UCSF Chimera, the activity
and stability of enzymes, both in wild-type and mu-
tant forms, can be comprehensively studied. SDM
in glycosyltransferases, a family of enzymes respon-
sible for glycosylation, has been suggested to assist
in the development of cancer drugs (Majeed et al.,
2024).
SDM is widely used for enzyme modification
to enhance catalytic efficiency. For example, terpe-
noids, known for their anti-inflammatory and anti-
tumor properties, and α-galactosidase, an enzyme
used in cancer diagnosis and therapy, have both
been improved through SDM to increase their cata-
lytic efficacy (Xu et al., 2014; Sarkar et al., 2022). Ad-
ditionally, a mutant superantigen,
SEC2(T20L/G22E), developed via SDM, has
demonstrated enhanced antitumor activity com-
pared to its native form in vitro (Wang et al., 2009).
A promising approach in cancer therapy, amino
acid deprivation therapy (AADT), inhibits cancer
cell proliferation by limiting amino acid availability
without affecting normal cells, which have lower
amino acid requirements. This makes AADT selec-
tive and non-toxic (Pokrovsky et al., 2022; Kumar et
al., 2022; Zhang et al., 2024).
Among targets for AADT, asparagine has
shown promise. L-asparaginase, an enzyme that hy-
drolyzes L-asparagine into L-aspartate and ammo-
nia, inhibits the availability of essential amino acids
in cancer cells, leading to cell death and reduced
proliferation (Butler et al., 2021). However, L-aspar-
aginase also accepts L-glutamine as a substrate,
which can lead to side effects like cerebral hemor-
rhage and neurological disorders due to decreased
glutamine levels (Van Trimpont et al., 2022; Feng-
min et al., 2023). Recent SDM efforts have improved
L-asparaginase’s activity, reducing its L-glutami-
nase activity and minimizing these side effects
(Zhang et al., 2024). Another recent breakthrough
includes the production of the anticancer com-
pound dehydroabietic acid through heterologous
expression in Saccharomyces cerevisiae via SDM (Ma
et al., 2024a). Nanobodies, traditionally produced
from camelid immune libraries, are widely used in
diagnostics and immunotherapy. However, due to
the complexity of animal-based procedures, recent
studies have utilized CDR grafting and SDM to pre-
pare genetically engineered nanobodies, such as
anti-CD20 nanobodies, for leukemia treatment.
SDM, when combined with other therapeutic tech-
niques, holds potential in treating cancer and tu-
mors (Gu et al., 2023; Heidari et al., 2024).
2. Enzyme Engineering/Therapy
Enzymes are essential biochemical catalysts,
facilitating specific reactions due to their protein-
based nature, which grants them high specificity to
distinguish substrates of similar structures. Their
catalytic efficiency is closely tied to the integrity of
their protein conformation (Tandon et al., 2021).
Mutations can compromise this efficiency, leading
to dysfunction in enzyme-related diseases, prompt-
ing the development of drugs targeting these com-
promised enzymes. In recent years, enzymes have
emerged as valuable tools for investigating and
treating various pathological conditions, including
cancer and metabolic deficiencies (Majeed et al.,
2024; Wang et al., 2024; Tvaroška, 2022). By utilizing
genetic engineering, enzymes can be modified to ac-
tivate under specific conditions, such as the hypoxic
or acidic environments found in tumor cells. Be-
sides creating new enzymes, existing enzymes can
be optimized for better activity through recombi-
nant DNA technology. This includes manipulating
molecular structures, altering amino acid sequences,
and introducing mutations via genetic engineering
(Majeed et al., 2024; Wang et al., 2024). These tech-
niques are crucial for enhancing desirable enzyme
traits, such as improving kinetic properties, increas-
ing thermal stability, adjusting optimal tempera-
tures, enhancing specificity, and eliminating allo-
steric regulations (Tandon et al., 2021).
Clinically and medically important com-
pounds are being produced by genetically introduc-
ing the gene encoding the desired product into var-
ious organisms, using genetic engineering tech-
niques (Fig. 2). This process has enabled the devel-
opment of several natural anticancer drugs through
enzyme engineering (Majeed et al., 2024; Wang et
al., 2024; Tvaroška, 2022). By bioengineering en-
zymes, researchers can assess the presence or ab-
sence of critical amino acids at catalytic sites, an ap-
proach made possible through methods such as do-
main swapping or site-directed mutagenesis (SDM).
These engineered enzymes are designed to target
and bind to specific structures and molecules found