THE UMMED SCHOOL, JODHPUR

AFFILIATION NO.-1730944

SESSION 2024-25

ASSESMENT PROJECT

TOPIC: A Thing of Beauty

SUBJECT: ENGLISH

CLASS: XII science

Submitted By
Raghav Sankhla

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 CANDIDATE'S DECLARATION

……………, a student of Class XII, hereby declare that the project work titled “Project Title” is
my own work and has been carried out under the guidance of Dr. Shikha Kapil. This project has
not been submitted to any other institution for any other degree or diploma.

I have acknowledged all sources of information used in the preparation of this project, and I have
adhered to the ethical guidelines and academic standards required for this work.

Candidate Name

Date………….
This is to certify that the above statement made by the candidate is correct to the best of my
knowledge.

Supervisor

Dr. Shikha Kapil

PGT, Biology
The Ummed School
Jodhpur

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 CERTIFICATE

This is to certify that ………. of class XII has successfully completed the project work on
Biology for class XII practical examination of the Central Board of Secondary Education in the
year 2024-25. It is further certified that this project is the individual work of the candidate.

Signature of Examiner

Signature of Supervisor

Signature of Principal

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 ACKNOWLEDGMENTS

I would like to express my deepest gratitude and heartfelt thanks to Dr. Shikha Kapil PGT at The
Ummed School, Jodhpur for believing in me. Thanks for investing your time, supervision,
patience, encouragement, critics, and refinement. Your concerned approaches not only uplift my
potential but also amplify my vision.
I am thankful to Ms. Ekta Baliya, (Principal, The Ummed School) to allow me to avail of
lab facilities. Thanks for the unlimited discussions and suggestions throughout my schooling
tenure.

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 TABLE OF CONTENTS

Candidate's declaration 2

Certificate 3

Acknowledgements 4

Abstract 5

Table of contents 6

List of figures 7

List of tables 8

List of abbreviations 9

1 Introduction 10
1.1 D-amino acids
11
1.2 Serine diastereomers
1.3 Synthesis of D-serine 12
2 Review of literature
13
2.1 Biochemistry of SR
2.2 SR Orthologues and Homologues 14
2.2.1 SR in mammals
15
2.3 Dual Metabolic Activity of SR
Summary

References

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 LIST OF FIGURES

Sr. No. Titles Page No.

2.1 Sequence alignment of human (hSR) (GI:11034785) and rat (rSR)

(GI:38454254).
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2.2 hSR (Dimeric form). 12

2.3 Diagrammatic representation of SR and D-serine-synthesis. 15

2.4 Dual reaction synthesized by SR (A) Racemization and (B) α-β elimination. 19

3.1 Derivatization of D-AAs using OPA/IBLC before the column. 36

4.1.1 The observed growth of the isolates after 48 hrs. of incubation in minimal 47
media broth.

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 LIST OF TABLES

Sr. No. Titles Page No.

2.1 Crystallographic structure of SR available in PDB. 13

2.2 Effects of various regulator proteins on SR. 16

2.3 Kinetic parameters of SRs isolated from different species. 21-22

2.4 The β-elimination activity of SR for the different substrates. 23

2.5 Summary of competitive SR inhibitors and their K1 value in µM. 25

4.1.1 Brief description of the sample and collecting sites from various districts of
northern India.
45

4.1.2 The recorded optimum temperature for Colonies grown on M9 minimal media
agar plates.
46

4.2.1 Recovery percentage (%) of a spiked sample of known concentrations. 52

4.3.1 Morphological and biochemical characteristics of the isolate A1C1. 57

4.8.1 The table contains details of organisms, such as species name, protein IDs,
protein length, and protein type.
78-84

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 LIST OF ABBREVIATIONS

(NH4)2SO4 Ammonium sulfate

ABOs Amyloid-β oligomers

AD Alzheimer's disease

Akt AK mouse plus Transforming or Thymoma

Alr Alanine racemase

ALS Amyotrophic Lateral Sclerosis

AMPA α-amino 3-hydroxy 5-methyl 4-isoxazole propionic acid

Asc-1 Alanine serine- cysteine-1

ASR Alanine serine racemase

ATP Adenosine triphosphate

AtSR Arabidopsis thaliana SR

BDNF Brain-derived neurotrophic factor

BLASTn Basic Local Alignment Search Tool for Nucleotides

Ca Calcium

CaCl2 Calcium chloride

CBB Coomassie brilliant blue

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 INTRODUCTION

1.1 D-amino acids

Louis Pasture was the first to observe optically active compounds in polarized light. These
compounds lack symmetrical planes and have non-superimposable mirror images. The
chirality of the compounds is the process of the rotation of plane-polarized light in clockwise
and anticlockwise directions. In the 1950s, chirality led to the discovery of amino acids and
proteins (1–3). Chiral active enantiomers of amino acids are found in both L & D-forms in
nature and are the basic structural unit of protein (4). Corrigan, in 1969, revealed the
importance of D-amino acids (D-AAs) such as D-asparagine, D-glutamine, D-serine, and D-
Alanine in the bacterial peptidoglycan (PG) cell wall (5). Two mechanisms drive d-AAs
synthesis: posttranslational modification with the help of enzymes in eukaryotes and non-
ribosomal peptide synthesis in prokaryotes. Various enzymes, such as amino acid racemases
and oxidases, are required for generating and degenerating D-AAs, respectively (6).
Bacteria utilize D-AAs for their various physiological activities, growth, and spore
germination (7–10). Other higher organisms, such as human beings also contain D-AAs
either in the free form or embedded in peptides and proteins. Reported D-AAs such as D-
serine and D-aspartate are abundant in the mammalian brain (11).
1.2 Serine diastereomers
In general, L-amino acids are significantly abundant in higher organisms which interferes
with determining D-amino acid molecules and, therefore, demands highly selective and
sensitive detection methods. It became possible after several decades with the discovery of
modern HPLC techniques that revealed significant levels of free, endogenous D-AAs in
mammals, specifically in the brain. Before detecting D-serine, Dunlop and co-workers found
abundant D-aspartic acid in various mammalian tissues (11–13). Although various D-AAs
are also present, their function is partially vague (14). D-serine and D-aspartic acid represent
5-30% of L-forms, and their role in mammalian tissues has been studied well (15). D-serine
is a well-studied and recognized regulator of various activities in the human nervous system
(16,17). D-serine acts as a co-agonist for the glycine modulatory site on N-methyl D-
aspartate receptors (NMDAr) for its activation (17,18). NMDARs belong to ligand-gated ion
channels and activate glutamate-mediated excitatory neurotransmission in the mammalian
nervous system. NMDAr is located in neurons and consists of different subunits NR1 and

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 NR2 to bind suitable co-against for its activation (19). In addition to glutamate, NMDAr
requires glycine or D-serine as a co-agonist for opening ion-gated channels (20,21). Until D-
serine detection in the mammalian brain, glycine was thought to be the chief co-agonist to
activate the ion influx (22,23). Later, D-serine proportion and binding affinity were found to
be more abundant for NMDAr than glycine (24,25). D-serine synthesis occurs in neurons,
predominantly regulates excitatory neurotransmission, and helps shape synaptic plasticity
(26,27,30–33).
Studies showed that enzymatic depletion of D-serine leads to a decrease in NMDAr
activation and NMDAr-mediated neuronal activities (25,30).
1.3 Synthesis of D-serine
Serine racemase (SR, EC.5.1.1.18) is a cytosolic and pyridoxal-5’-phosphate (PLP)
dependent enzyme responsible for the synthesis of D-serine in the mammalian brain (28,31–
33). Besides SR-mediated D-serine production, no other pathways report its synthesis
(31,32). Before SR detection, food, and gut microbiome were considered significant sources
of D-seine content in mammalian tissues. The statement was justified by the studies reporting
D-serine transportation to the mammalian brain through the blood-brain barrier (12,34,35).
1.4 Degradation of D-serine
The discovery of D-amino acid oxidase (DAAO), an enzyme responsible for D-serine’s
degradation, has unveiled its importance in the mammalian brain. DAAO is a Flavin-adenine
dinucleotide (FAD) enzyme first identified in 1935. DAAO catalyzes the oxidative
deamination of D-serine and converts it into hydroxy pyruvate (43-45). Further, the role of
DAAO in NMDAr-related neuropathological conditions is also studied. With the discovery
of the G72 gene, a potential interaction partner of DAAO and associated with schizophrenia,
many studies have defined the role of DAAO in schizophrenia (37,38). The experiments
conducted with DAAO-deficient mice have strengthened the previous statement (39,40).
Other than DAAO, SR is responsible for D-serine degradation itself. SR produces pyruvate
from D-serine through α-β elimination of water molecules, which is its second catalytic
activity (41,42) (for further details, see Chapter 2, Section 2.1.2).
1.5 Role of D-serine
In the past decade, many studies reported the role of glial cells and neurons in synaptic
plasticity in the mammalian CNS. A synapse is a junction surrounded by glial cells where

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neurotransmitter transportation occurs between neurons. Astrocytes, a subset of glial cells,
transmit several neurotransmitters such as nucleotides, nitric oxide (NO), amino acids such as
D-serine and glutamate, etc. (43–45). These compounds are essential to regulate overall
functions and the development of neuronal tissues (46,47). To this, these neurotransmitters
are supposed to be released and removed efficiently from the synapse. Neurotransmitters
accumulate in secretory vesicles and transport through the cell membrane into synapses upon
Ca2+ stimulation (48,49). D-serine and glutamate also use the same vesicles for transportation
into synapses through amino acid transporters on the neuronal cell membrane (48,50). D-
serine binds efficiently to alanine serine cysteine-1 (Asc-1) transporters and releases into
synaptic space (50–52). D-serine attaches to the receptors in postsynaptic neurons to regulate
synaptic plasticity after it releases from presynaptic neurons.
D-serine has biological importance, and its biosynthetic pathway is significantly studied.
SR (EC.5.1.1.18), a multifunctional and PLP-dependent enzyme, is responsible for its
synthesis. SR synthesizes D-serine from L-serine through the racemization process (53). SR
genes are present in prokaryotes and eukaryotes and are further studied for enzymatic
properties/activities (54). Howbeit, our knowledge in the case of prokaryotic SRs is still
limited. For instance, the synthesis of D-serine in marine heterotrophs is unstable and inhibits
the bacterial growth of Roseobacter litoralis Och 149 (55).
Few bacterial strains synthesize D-serine in the environment (55–58). Moreover, in
our environment, 3-7% of total serine is present in soil (59), 5-10% of total serine in
rivers (60), and 4% of total serine in lakes (61). Interestingly, serine diastereomers are
also present in antimicrobial peptides (2-3%) (62), siderophores (63), bacterial lipopeptide
biosurfactants (64), and bacterial PG (65).
Molecular/genetic markers have enormous potential for exploring the emergence and
transformations of organic material. Still, it is essential to understand biomarkers' biological
sources and diagnostic reactivity for quantitative applications. D-AAs are crucial components
of the bacterial cell walls, making them valuable molecular/genetic markers for bacterial-
derived organic material in the surrounding environment (58). In recent years, evidence has
reported free D-AAs such as D-aspartate and D-serine in prokaryotes and eukaryotes (54).

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