1. Introduction
1.1 Characterization of Glutamate Decarboxylase (GAD)
Glutamate decarboxylase (GAD) (E.C.4.1.1.15) is classified under enzymatic category of carboxy-lyases [1-3]. GAD catalytic activity involves solely removal of carboxyl group from L-glutamate/glutamic acid, follows by the formation of gamma-aminobutyric acid (GABA) and carbon dioxide (CO2) [3,4] (Figure 1, Appendices 8-4, 8-6, 8-9). To accomplish this catalytic reaction, GAD requires pyridoxal phosphate (PLP) as cofactor, an active form of Vitamin B6 [5].
1.2 PLP as a Cofactor of GAD
Pyridoxal phosphate (PLP) is an organic chemical compound that commonly known as active form of vitamin B6. It is strictly required in all transamination reactions, and in several amino acid decarboxylation such as GAD and tyrosine decarboxylase [5]. Without PLP, GAD is an apoenzyme.
By forming Schiff-base, carbon-nitrogen double bonds between aldehyde group of PLP with the ε-amino group on a specific lysine, GAD turns out be catalytically active holoenzyme [6].
Unicellular prokaryotic microbes such as E. coli and B. subtilis are capable of synthesizing vitamin B6 as a precursor of PLP in their metabolic pathway [7]. In order to achieve maximal production of GABA in industrial
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bioreactor, GABA production should consider microbial synthesis of PLP concurrent with that of GAD reaction.
1.3 GAD in Major Organisms
Glutamate decarboxylase is an essential enzyme widely distributed among eukaryotes and prokaryotes. GAD can be found in vast variety of organisms, ranging from unicellular prokaryotes such as gram-negative bacteria E. coli and Francisella tularensis, thermophilic archaea such as Thermococcus kodakarensis and P. abyssi, to complex plants and animals [8]
(Appendix 8-9). There are usually several isoforms of GADs present in most of the organisms. For example, GADs of Homo sapiens and Mus musculus have two isoforms [5], whereas five GAD isoforms were identified in Arabidopsis thaliana [9]. Even though these isoforms have similar function, the quantity and catalytic efficiency of each isoform that presents within an organism is very distinct from one another.
GAD activity of mesophilic organism catalyzes substrate to form end product optimally at moderate temperature of natural habitat where the organism usually survives in. However, hyperthermophilic archaeon thrives in hydrothermal vent where temperature can easily hit boiling point of water [22]. Thus, we expect GAD of hyperthermophilic archaeon possesses activity even at high temperature that is no less than mesophilic GADs which makes GAD of hyperthermophilic archaeon has superior catalytic temperature than that of mesophilic GADs. This is because high temperature
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tends to denature most enzymes [80,81] including mesophilic GADs, but not hyperthermophilic archaeon.
Organism GAD optimum catalytic temperature Escherichia coli GADb
GADs’ optimal temperature of various organisms.
1.4 The biological function of GABA in Mammals and Plant
As a major inhibitory neurotransmitter in mammalian brain and central nervous system (CNS), GABA is mainly formed in situ, nearby GABAergic synapses of nerve cells [10]. Upon stimulation, GABA is first released from vesicle to synaptic cleft of synapse. Then, GABAA and GABAB receptors [11,12] respond upon GABA binding and hence causes4
opening of ion channels, resulting hyperpolarization. GABA acts on its receptors would have typical relaxing and anti-anxiety effects [10]. In contrast with GABA, glutamate acts on nervous receptors to exhibit excitatory effect [13]. In plant such as Arabidopsis thaliana, GABA assumes the role of signalling molecule that reacts toward stress response, cellular pH homeostasis and pest invasion [87]. In prokaryotic bacteria such as E. coli and Listeria monocytogenes, GAD system confers acid resistance by consuming cytosolic hydrogen ions to produce GABA from L-glutamate and thus, increasing intracellular pH value [77,78].
1.5 GABA Deficiency Related Diseases
Throughout the years, many studies indicated GAD disorder may lead to health deterioration, for instance, type-1 diabetes mellitus, epilepsy, bipolar disorder and Parkinson’s disease [14,15]. According to previous medical research, autoimmunity targeting GADs in human body leads to type-1 diabetes mellitus [16], moreover, reduced GAD genes expression is closely associated with schizophrenia and bipolar disorder as well [13,14].
Currently, there are only two GABA analogues, pregabalin and gabapentin (Appendix 8-9), approved and produced as anticonvulsant, antiepilepticor and antidepressant drugs. They are marketed by pharmaceutical companies as medication to treat the diseases produced by disruption of GAD [17,18]. Therefore, there is huge potential in GABA production via enzymatic catalysis, utilizing GAD as biocatalyst.
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1.6 GABA as Industrial and Pharmaceutical Materials
GABA can be taken orally to relieve anxiety, improving moods, reducing premenstrual syndrome symptoms (PMS) and treating attention deficit-hyperactivity disorder (ADHD). It is also prescribed for promoting lean muscle growth, fat burning, stabilizing blood pressure, and relieving pain. Hence, GABA itself has huge market potential in medical industry and production of GABA by the mean of GAD enzyme would have value added effect because it does not require or generate harmful and deteriorating organic chemicals during the production.
Apart from medication, GABA can be utilized by polymer and pharmaceutical industries as it is a precursor of 2-pyrrolidone [20] organic compound, a monomer of Nylon 4 polymer (Appendix 8-10). Unlike other chemically synthesized polymers, the advantage of Nylon 4 over others is that it is biodegradable polymer [21]. Consequently, Nylon 4 could be degraded by Pseudomonas species [21] and considered an environmental-friendly material. The biodegradation produces GABA as byproduct, which could be further catabolised by other microbes into simpler molecules.
There are variety of pharmaceutical drugs that derived from 2-pyrrolidone (Appendix 8-10) as well. Such drugs are cotinine, doxapram, piracetam and so forth [20]. Therefore, mass production of GABA has enormous commercial value and GAD catalytic conversion of L-glutamate to GABA will be deemed as green technology as compared to chemically produced GABA.
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1.7 Biochemical Morphology of P. abyssi GE5 and Its GAD
Pyrococcus abyssi GE5 (P.abyssi) is a model of hyperthermophilic archaeon [22] discovered in the North Fiji Basin of pacific ocean. It survives under deep-sea hydrothermal vent whose temperature can easily strike over hundreds degree Celsius (°C). Complete genomic sequence of P. abyssi was published in 2001 [88]. Its genomic sequence is roughly 1.7 million bp and has 1878 estimated genes [88]. P. abyssi indicated as hyperthermophile due to its tolerance towards extreme temperature [22]. Besides that, P. abyssi is categorized under gram-negative, anaerobic and sulphur-metabolizing archeon with an optimal surviving temperature of 96°C [22]. Theoretically, entire cellular structures and components of P. abyssi including its enzymes should be marvellous at resisting heat denaturation under high temperature where mesophilic microbes could not. So, we hypothesized GAD of P.abyssi should remain catalytically active at high temperature and it functions as acid tolerance system, which is supposedly similar to that of E. coli and Listeria monocytogenes GAD systems[77,78].
To conduct an experiment so that the hypothesis drawn can be validated, a putative cDNA encoding GAD of P.abyssi was cloned into expression vector. Then, vector carrying the gene would be transformed into BL-21 (DE3) competent cell expression system. This expression system requires inducers to promote highly effective synthesis of recombinant enzyme at huge quantity, so that there is sufficient rPaGAD (recombinant P.
abyssi GAD) for catalytic analysis.
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1.8 Analysis of GABA as End Product
Formation of GABA cannot be detected directly using UV/Vis detectors. Because lacking of benzyl group that emits visible light upon UV irradiation, GABA is colorless no matter dissolved in water or organic solvent [23]. Thus, GABA must be derivatized into another compound that possesses optical density or excitable via irradiation so that it is distinguishable by detector. This step is a crucial part as it affects latter experimental progression whenever quantification of GABA is necessary. A simple way to derivatize GABA is to mix briefly with OPA and MPA in basic buffer [42,43,57] (Appendix 8-11). OPA contains a benzyl group, it illuminates blue spectrum upon UV irradiation [42]. This protocol would be further explained in section 3.1.17 of materials and methods
1.9 Futuristic Application of Pyrococcus Abyssi’s GAD
GAD of P. abyssi has the potential to be utilized by commercial industries as biocatalysis to produce valuable GABA because of its tolerance towards high temperature and still remains catalytically active. These properties are advantageous in bioreactor because reaction that takes place under high temperature prevents growth of polluting microbes [24] and hence, securing high quality production of GABA as end product. Apart from that, efficiency of GABA production tends to increase corresponding to higher temperature as greater kinetic energy [25] engages more substrate-enzyme interaction at shorter period.
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Employing P. abyssi GAD as biocatalyst to product GABA could avoid pollution to a certain extent. Since origin of GAD belongs to unicellular microbe and it is proteinaceous, it is expected to be biodegradable. Besides that, biocatalyst such as GAD does not require toxic and hazardous chemicals as cofactors during biochemical transformation, and thus reducing potential pollutants being released to the environment or increasing cost of waste treatment.
1.10 Phylogenetic Study on Divergence and Evolution of Glutamate Decarboxylases of Various Organisms
Phylogenetic study was employed to study evolution between various GADs from different organisms. The deduced amino acid sequence of rPaGAD with GAD from other species in database uses the Molecular Evolutionary Genetics Analysis (MEGA) software to generate phylogenetic tree of GADs [26]. The similar the sequences, the closer they are allocated together. In contrast, GADs of distinct species should be distant.
Apart from phylogenetic study, multiple sequence alignment platform, Cluster Omega [27] was used to cluster highly conversed amino acids within sequences of GAD. Then, these conserved regions would be tabulated across organisms of different biochemical morphology, showing the critical parts of GADs which attributed as active sites.
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EcGADβ PaGAD AtGAD1 AfGAD ToGAD TkGAD FtGAD
EcGADβ 100 24 45 59 25 28 57
PaGAD 100 25 26 79 76 25
AtGAD1 100 47 25 27 48
AfGAD 100 25 28 69
ToGAD 100 85 26
TkGAD 100 29
FtGAD 100
Pairwise identities of GADs between various organisms.
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