Browsing by Author "Tinikul R."
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Item Metadata only A high catalytic efficiency and chemotolerant formate dehydrogenase from Bacillus simplex(2024-01-01) Boonkumkrong R.; Chunthaboon P.; Munkajohnpong P.; Watthaisong P.; Pimviriyakul P.; Maenpuen S.; Chaiyen P.; Tinikul R.; Boonkumkrong R.; Mahidol UniversityNAD+-dependent formate dehydrogenase (FDH) catalyzes the conversion of formate and NAD+ to produce carbon dioxide and NADH. The reaction is biotechnologically important because FDH is widely used for NADH regeneration in various enzymatic syntheses. However, major drawbacks of this versatile enzyme in industrial applications are its low activity, requiring its utilization in large amounts to achieve optimal process conditions. Here, FDH from Bacillus simplex (BsFDH) was characterized for its biochemical and catalytic properties in comparison to FDH from Pseudomonas sp. 101 (PsFDH), a commonly used FDH in various biocatalytic reactions. The data revealed that BsFDH possesses high formate oxidizing activity with a kcat value of 15.3 ± 1.9 s−1 at 25°C compared to 7.7 ± 1.0 s−1 for PsFDH. At the optimum temperature (60°C), BsFDH exhibited 6-fold greater activity than PsFDH. The BsFDH displayed higher pH stability and a superior tolerance toward sodium azide and H2O2 inactivation, showing a 200-fold higher Ki value for azide inhibition and remaining stable in the presence of 0.5% H2O2 compared to PsFDH. The application of BsFDH as a cofactor regeneration system for the detoxification of 4-nitrophenol by the reaction of HadA, which produced a H2O2 byproduct was demonstrated. The biocatalytic cascades using BsFDH demonstrated a distinct superior conversion activity because the system tolerated H2O2 well. Altogether, the data showed that BsFDH is a robust enzyme suitable for future application in industrial biotechnology.Item Metadata only A Multiplexed Cas13-Based Assay with Point-of-Care Attributes for Simultaneous COVID-19 Diagnosis and Variant Surveillance(2023-04-01) Patchsung M.; Homchan A.; Aphicho K.; Suraritdechachai S.; Wanitchanon T.; Pattama A.; Sappakhaw K.; Meesawat P.; Wongsatit T.; Athipanyasilp A.; Jantarug K.; Athipanyasilp N.; Buahom J.; Visanpattanasin S.; Niljianskul N.; Chaiyen P.; Tinikul R.; Wichukchinda N.; Mahasirimongkol S.; Sirijatuphat R.; Angkasekwinai N.; Crone M.A.; Freemont P.S.; Joung J.; Ladha A.; Abudayyeh O.; Gootenberg J.; Zhang F.; Chewapreecha C.; Chanarat S.; Horthongkham N.; Pakotiprapha D.; Uttamapinant C.; Mahidol UniversityPoint-of-care (POC) nucleic acid detection technologies are poised to aid gold-standard technologies in controlling the COVID-19 pandemic, yet shortcomings in the capability to perform critically needed complex detection - such as multiplexed detection for viral variant surveillance - may limit their widespread adoption. Herein, we developed a robust multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)-based detection using LwaCas13a and PsmCas13b to simultaneously diagnose severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and pinpoint the causative SARS-CoV-2 variant of concern (VOC) - including globally dominant VOCs Delta (B.1.617.2) and Omicron (B.1.1.529) - all the while maintaining high levels of accuracy upon the detection of multiple SARS-CoV-2 gene targets. The platform has several attributes suitable for POC use: premixed, freeze-dried reagents for easy use and storage; convenient direct-to-eye or smartphone-based readouts; and a one-pot variant of the multiplexed detection. To reduce reliance on proprietary reagents and enable sustainable use of such a technology in low- and middle-income countries, we locally produced and formulated our own recombinase polymerase amplification reaction and demonstrated its equivalent efficiency to commercial counterparts. Our tool - CRISPR-based detection for simultaneous COVID-19 diagnosis and variant surveillance that can be locally manufactured - may enable sustainable use of CRISPR diagnostics technologies for COVID-19 and other diseases in POC settings.Item Metadata only Biochemical and structural characterization of meningococcal methylenetetrahydrofolate reductase(2023-06-01) Pantong W.; Pederick J.L.; Maenpuen S.; Tinikul R.; Jayapalan J.J.; Jovcevski B.; Wegener K.L.; Bruning J.B.; Salaemae W.; Mahidol UniversityMethylenetetrahydrofolate reductase (MTHFR) is a key metabolic enzyme in colonization and virulence of Neisseria meningitidis, a causative agent of meningococcal diseases. Here, the biochemical and structural properties of MTHFR from a virulent strain of N. meningitidis serogroup B (NmMTHFR) were characterized. Unlike other orthologs, NmMTHFR functions as a unique homohexamer, composed of three homo-dimerization partners, as shown in our 2.7 Å resolution crystal structure. Six active sites were formed solely within monomers and located away from the oligomerization interfaces. Flavin adenine dinucleotide cofactor formed hydrogen bonds with conserved sidechains, positioning its isoalloxazine ring adjacent to the overlapping binding sites of nicotinamide adenine dinucleotide (NADH) coenzyme and CH2-H4folate substrate. NmMTHFR utilized NADH (Km = 44 μM) as an electron donor in the NAD(P)H-CH2-H4folate oxidoreductase assay, but not nicotinamide adenine dinucleotide phosphate (NADPH) which is the donor required in human MTHFR. In silico analysis and mutagenesis studies highlighted the significant difference in orientation of helix α7A (Phe215–Thr225) with that in the human enzyme. The extended sidechain of Met221 on helix α7A plays a role in stabilizing the folded structure of NADH in the hydrophobic box. This supports the NADH specificity by restricting the phosphate group of NADPH that causes steric clashes with Glu26. The movement of Met221 sidechain allows the CH2-H4folate substrate to bind. The unique topology of its NADH and CH2-H4folate binding pockets makes NmMTHFR a promising drug target for the development of new antimicrobial agents that may possess reduced off-target side effects.Item Metadata only Biochemical characterization and inhibitor potential of African swine fever virus thymidine kinase(2025-03-01) Rasri N.; Kornyanee C.; Srisanga K.; Nutho B.; Chanarat S.; Kuhaudomlarp S.; Tinikul R.; Pakotiprapha D.; Rasri N.; Mahidol UniversityAfrican Swine Fever (ASF) is a highly contagious disease affecting both domestic pigs and wild boars. In domestic pigs, ASF is a rapidly-progressing disease with a mortality rate reaching 100 %, causing tremendous economic loss in affected areas. ASFV is caused by African Swine Fever Virus (ASFV), which is a large, enveloped double-stranded DNA virus belonging to the Asfarviridae family. ASFV has a remarkably large genome size that encodes more than 150 open reading frames. Among the virally encoded enzymes, thymidine kinase (ASFV-TK) has been shown to be critical for the efficient replication and virulence of ASFV. Here, we report the bioinformatics analysis and biochemical characterization of ASFV-TK. Amino acid sequence analysis revealed that ASFV-TK can be classified as a type II thymidine kinase. Kinetics characterization revealed a maximum velocity (Vmax) and Michaelis constants (Km) that are within the same range as previously characterized type II enzymes. ASFV-TK is competitively inhibited by the feedback inhibitor thymidine 5′-triphosphate and can use 3′-azido-3′-deoxythymidine (AZT) as a substrate with kinetics parameters comparable to those obtained with natural substrates, suggesting that nucleosides and nucleotide analogs could be explored as anti-ASFV agents.Item Metadata only Bridging the Gap: Can COVID-19 Research Help Combat African Swine Fever?(2023-09-01) Pakotiprapha D.; Kuhaudomlarp S.; Tinikul R.; Chanarat S.; Mahidol UniversityAfrican swine fever (ASF) is a highly contagious and economically devastating disease affecting domestic pigs and wild boar, caused by African swine fever virus (ASFV). Despite being harmless to humans, ASF poses significant challenges to the swine industry, due to sudden losses and trade restrictions. The ongoing COVID-19 pandemic has spurred an unparalleled global research effort, yielding remarkable advancements across scientific disciplines. In this review, we explore the potential technological spillover from COVID-19 research into ASF. Specifically, we assess the applicability of the diagnostic tools, vaccine development strategies, and biosecurity measures developed for COVID-19 for combating ASF. Additionally, we discuss the lessons learned from the pandemic in terms of surveillance systems and their implications for managing ASF. By bridging the gap between COVID-19 and ASF research, we highlight the potential for interdisciplinary collaboration and technological spillovers in the battle against ASF.Item Metadata only Detection of cellular metabolites by redox enzymatic cascades(2022-06-01) Tinikul R.; Trisrivirat D.; Chinantuya W.; Wongnate T.; Watthaisong P.; Phonbuppha J.; Chaiyen P.; Mahidol UniversityDetection of cellular metabolites that are disease biomarkers is important for human healthcare monitoring and assessing prognosis and therapeutic response. Accurate and rapid detection of microbial metabolites and pathway intermediates is also crucial for the process optimization required for development of bioconversion methods using metabolically engineered cells. Various redox enzymes can generate electrons that can be employed in enzyme-based biosensors and in the detection of cellular metabolites. These reactions can directly transform target compounds into various readout signals. By incorporating engineered enzymes into enzymatic cascades, the readout signals can be improved in terms of accuracy and sensitivity. This review critically discusses selected redox enzymatic and chemoenzymatic cascades currently employed for detection of human- and microbe-related cellular metabolites including, amino acids, d-glucose, inorganic ions (pyrophosphate, phosphate, and sulfate), nitro- and halogenated phenols, NAD(P)H, fatty acids, fatty aldehyde, alkane, short chain acids, and cellular metabolites.Item Metadata only Differential expression of neuropeptide F during embryogenesis, and its promoting effect on embryonic development of the freshwater prawn, Macrobrachium rosenbergii(2022-06-30) Tinikul Y.; Tinikul R.; Poljaroen J.; Sobhon P.; Mahidol UniversityIn the present study, we examined differential expression of Macrobrachium rosenbergii neuropeptide F (MrNPF) transcripts, and studied the presence of MrNPF during embryonic stages. We further investigated the effect for MrNPF on embryonic development of M. rosenbergii. By using RT-qPCR, the MrNPF mRNA levels showed a gradual increase from the early embryonic stages to the late embryonic stages. This coincides with initial expression of MrNPF immunoreactivity (MrNPF-ir) in the early embryonic stages and later strong MrNPF-ir in the late embryonic stages. Treating with MrNPF at doses of 10−6 and 10−7 mol/prawn into the female prawns significantly shortened the lengths of embryonic periods, exhibited faster attainment of each embryonic stage, and increased the number of proliferative cells, compared with the control groups (P < 0.05). In addition, the percent of fertilization was higher in the MrNPF-treated group at a dose of 10−6 mol/prawn, compared with the control groups (P < 0.05). The mean egg size of MrNPF-treated groups at doses of 10−6 and 10−7 mol/prawn also was slightly larger than that of the control groups. Following treatment with three doses of MrNPF, developing embryos also showed normal development in terms of eye pigmentation and differentiation, appearance of a beating heart, appearance of oily yolk, formation of the abdomen, and folding of the telson. Taken together, our results provide new insight into the potential role of MrNPF in the embryonic development of M. rosenbergii; this knowledge may be useful in improving methods of seed production in this economically-important crustacean species.Item Metadata only Differential expression of neuropeptide F in the digestive organs of female freshwater prawn, Macrobrachium rosenbergii, during the ovarian cycle(2024-01-01) Vetkama W.; Tinikul R.; Sobhon P.; Tinikul Y.; Vetkama W.; Mahidol UniversityNeuropeptide F is a key hormone that controls feeding in invertebrates, including decapod crustaceans. We investigated the differential expression of Macrobrachium rosenbergii neuropeptide F (MrNPF) in the digestive organs of female prawns, M. rosenbergii, during the ovarian cycle. By using RT-qPCR, the expression of MrNPF mRNA in the esophagus (ESO), cardia (CD), and pylorus (PY) of the foregut (FG) gradually increased from stage II and peaked at stage III. In the midgut (MG), hindgut (HG), and hepatopancreas (HP), MrNPF mRNA increased from stage I, reaching a maximal level at stage II, and declined by about half at stages III and IV (P < 0.05). In the ESO, CD, and PY, strong MrNPF-immunoreactivities were seen in the epithelium, muscle, and lamina propria. Intense MrNPF-ir was found in the MG cells and the muscular layer. In the HG, MrNPF-ir was detected in the epithelium of the villi and gland regions, while MrNPF-ir was also more intense in the F-, R-, and B-cells in the HP. However, we found little colocalization between the MrNPF and PGP9.5/ChAT in digestive tissues, implying that most of the positive cells might not be neurons but could be digestive tract-associated endocrine cells that produce and secrete MrNPF to control digestive organ functions in feeding and utilizing feed. Taken together, our first findings indicated that MrNPF was differentially expressed in digestive organs in correlation with the ovarian cycle, suggesting an important link between MrNPF, the physiology of various digestive organs in feeding, and possibly ovarian maturation in female M. rosenbergii.Item Metadata only High sensitivity and low-cost flavin luciferase (FLUXVc)-based reporter gene for mammalian cell expression(2023-05-01) Phonbuppha J.; Tinikul R.; Ohmiya Y.; Chaiyen P.; Mahidol UniversityLuciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase from Vibrio campbellii (Vc) for Mammalian Cell Expression (FLUXVc) by engineering luciferase from V. campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells which can improve the bioluminescence light output by >400-fold as compared to the nonengineered version. We found that the FLUXVc reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUXVc/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We also demonstrated that FLUXVc can be used for testing inhibitors of the NF-κB signaling pathway. Collectively, our results provide an optimized method for using the more economical flavin-dependent luciferase in mammalian cells.Item Metadata only In Silico and In Vitro Potential of FDA-Approved Drugs for Antimalarial Drug Repurposing against Plasmodium Serine Hydroxymethyltransferases(2023-01-01) Mee-Udorn P.; Phiwkaow K.; Tinikul R.; Sanachai K.; Maenpuen S.; Rungrotmongkol T.; Mahidol UniversityMalaria has spread in many countries, with a 12% increase in deaths after the coronavirus disease 2019 pandemic. Malaria is one of the most concerning diseases in the Greater Mekong subregion, showing increased drug-resistant rates. Serine hydroxymethyltransferase (SHMT), a key enzyme in the deoxythymidylate synthesis pathway, has been identified as a promising antimalarial drug target due to its conserved folate binding pocket. This study used a molecular docking approach to screen 2509 US Food and Drug Administration (FDA)-approved drugs against seven Plasmodium SHMT structures. Eight compounds had significantly lower binding energies than the known SHMT inhibitors pyrazolopyran(+)-86, tetrahydrofolate, and antimalarial drugs, ranging from 4 to 10 kcal/mol. Inhibition assays testing the eight compounds against Plasmodium falciparum SHMT (PfSHMT) showed that amphotericin B was a competitive inhibitor of PfSHMT with a half-maximal inhibitory concentration (IC50) of 106 ± 1 μM. Therefore, a 500 ns molecular dynamics simulation of PfSHMT/PLS/amphotericin B was performed. The backbone root-mean-square deviation of the protein-ligand complex indicated the high complex stability during simulations, supported by its radius of gyration, hydrogen-bond interactions, and number of atom contacts. The appreciable binding affinity of amphotericin B for PfSHMT was indicated by their solvated interaction energy (−11.15 ± 0.09 kcal/mol) and supported by strong ligand-protein interactions (≥80% occurrences) with its essential residues (i.e., Y78, K151, N262, F266, and V365) predicted by pharmacophore modeling and per-residue decomposition free energy methods. Therefore, our findings identify a promising new PfSHMT inhibitor, albeit with less inhibitory activity, and suggest a core structure that differs from that of previous SHMT inhibitors, thus being a rational approach for novel antimalarial drug design.Item Metadata only Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades(2022-04-11) Watthaisong P.; Kamutira P.; Kesornpun C.; Pongsupasa V.; Phonbuppha J.; Tinikul R.; Maenpuen S.; Wongnate T.; Nishihara R.; Ohmiya Y.; Chaiyen P.; Mahidol UniversityD-Luciferin (D-LH2), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method using enzymatic reactions (HELP, HadA Enzyme for Luciferin Preparation) to convert 19 phenolic derivatives to 8 D-LH2 analogues with ≈51 % yield. The method can synthesize the novel 5′-methyl-D-LH2 and 4′,5′-dimethyl-D-LH2, which have never been synthesized or found in nature. 5′-Methyl-D-LH2 emits brighter and longer wavelength light than the D-LH2. Using HELP, we further developed LUMOS (Luminescence Measurement of Organophosphate and Derivatives) technology for in situ detection of organophosphate pesticides (OPs) including parathion, methyl parathion, EPN, profenofos, and fenitrothion by coupling the reactions of OPs hydrolase and Fluc. The LUMOS technology can detect these OPs at parts per trillion (ppt) levels. The method can directly detect OPs in food and biological samples without requiring sample pretreatment.Item Metadata only Mangiferin is a new potential antimalarial and anticancer drug for targeting serine hydroxymethyltransferase(2023-09-01) Maenpuen S.; Mee-udorn P.; Pinthong C.; Athipornchai A.; Phiwkaow K.; Watchasit S.; Pimviriyakul P.; Rungrotmongkol T.; Tinikul R.; Leartsakulpanich U.; Chitnumsub P.; Mahidol UniversityMangiferin, a polyphenolic xanthone glycoside found in various botanical sources, including mango (Mangifera indica L.) leaves, can exhibit a variety of bioactivities. Although mangiferin has been reported to inhibit many targets, none of the studies have investigated the inhibition of serine hydroxymethyltransferase (SHMT), an attractive target for antimalarial and anticancer drugs. SHMT, one of the key enzymes in the deoxythymidylate synthesis cycle, catalyzes the reversible conversion of l-serine and (6S)-tetrahydrofolate (THF) into glycine and 5,10-methylene THF. Here, in vitro and in silico studies were used to probe how mangiferin isolated from mango leaves inhibits Plasmodium falciparum and human cytosolic SHMTs. The inhibition kinetics at pH 7.5 revealed that mangiferin is a competitive inhibitor against THF for enzymes from both organisms. Molecular docking and molecular dynamic (MD) simulations demonstrated the inhibitory effects of the deprotonated forms of mangiferin, specifically the C6-O- species and its resonance C9-O- species appearing at pH 7.5, combined with two docked poses, either a xanthone or glucose moiety, placed inside the THF-binding pocket. The MD analysis revealed that both C6-O- and its resonance-stabilized C9-O- species can favorably bind to SHMT in a similar fashion to THF, supporting the THF competitive inhibition of mangiferin. In addition, characterization of the proton dissociation equilibria of isolated mangiferin revealed that only three hydroxy groups of the xanthone moiety, C6-OH, C3-OH, and C7-OH, underwent varying degrees of deprotonation with pKa values of 6.38 ± 0.11, 8.21 ± 0.35, and 12.37 ± 0.30, respectively, while C1-OH remained protonated. Altogether, our findings demonstrate a new bioactivity of mangiferin and provide the basis for the future development of mangiferin as a potent antimalarial and anticancer drug.Item Metadata only Mechanisms and applications of bacterial luciferase and its auxiliary enzymes(2025-03-01) Kantiwiriyawanitch C.; Leartsakulpanich U.; Chaiyen P.; Tinikul R.; Kantiwiriyawanitch C.; Mahidol UniversityBacterial luciferase (LuxAB) catalyzes the conversion of reduced flavin mononucleotide (FMNH⁻), oxygen, and a long-chain aldehyde to oxidized FMN, the corresponding acid and water with concomitant light emission. This bioluminescence reaction requires the reaction of a flavin reductase such as LuxG (in vivo partner of LuxAB) to supply FMNH⁻ for the LuxAB reaction. LuxAB is a well-known self-sufficient luciferase system because both aldehyde and FMNH⁻ substrates can be produced by the associated enzymes encoded by the genes in the lux operon, allowing the system to be auto-luminous. This makes it useful for in vivo applications. Structural and functional studies have long been performed in efforts to gain a better understanding of the LuxAB reaction. Recently, continued exploration of the LuxAB reaction have elucidated the mechanisms of C4a-hydroperoxyflavin formation and identified key catalytic residues such as His44 that facilitates the generation of flavin intermediates important for light generation. Advancements in protein engineering and synthetic biology have improved the bioluminescence properties of LuxAB. Various applications of LuxAB for bioimaging, bioreporters, biosensing in metabolic engineering and real-time monitoring of aldehyde metabolites in biofuel production pathways have been developed during the last decade. Challenging issues such as achieving red-shifted emissions, optimizing the signal intensity and identifying mechanisms related to the generation of light-emitting species remain to be explored. Nevertheless, LuxAB continues to be a promising tool for diverse biotechnological and biomedical applications.Item Metadata only Mechanistic roles of the neighbouring cysteine in enhancing nucleophilicity of catalytic residue in a two-cysteine succinic semialdehyde dehydrogenase(2023-05-01) Paladkong T.; Pimviriyakul P.; Phonbuppha J.; Maenpuen S.; Chaiyen P.; Tinikul R.; Mahidol UniversitySuccinic semialdehyde dehydrogenase (SSADH) catalyses the conversion of succinic semialdehyde into succinic acid and two electrons are transferred to NAD(P)+ to yield NAD(P)H. Our previous work has already reported the catalytic role of Cys289 of two-cysteine SSADH from Acinetobacter baumannii (AbSSADH). However, the mechanistic role of the neighbouring conserved Cys291 and Glu255 remains unexplored. In this study, the functional roles of Cys291 and Glu255 in AbSSADH catalysis have been characterized. Results demonstrated that the E255A activity was almost completely lost, ~ 7000-fold lower than the wild-type (WT), indicating that Glu255 is very crucial and directly involved in AbSSADH catalysis. However, the C291A and C291S variants activity and catalytic turnover (kcat) decreased ~ 2-fold and 9-fold respectively. To further characterize the functional roles of Cys291, we employed two pH-dependent methods; pre-steady-state burst amplitude and NADP–enzyme adduct formation. The results showed that the pKa values of catalytic Cys289 measured for the WT and C291A reactions were 7.8 and 8.7–8.8, respectively, suggesting that Cys291 can lower the pKa of Cys289 and consequently trigger the deprotonation of a Cys289 thiol. In addition, the Cys291 also plays a role in disulfide/sulfhydryl redox regulation for AbSSADH activity. Hence, we demonstrated for the first time the dual functions of Cys291 in enhancing the nucleophilicity of the catalytic Cys289 and regulating a disulfide/sulfhydryl redox switch for AbSSADH catalysis. The mechanistic insights into the nucleophilicity enhancement of the catalytic cysteine of AbSSADH might be applicable to understanding how the microenvironment increases cysteine reactivity in other enzymes in the aldehyde dehydrogenase superfamily.Item Metadata only Morphological and histochemical characteristics of the foregut, midgut, and hindgut, and their alterations during ovarian development in female freshwater prawn, Macrobrachium rosenbergii(2025-01-01) Vetkama W.; Tinikul R.; Sobhon P.; Tinikul Y.; Vetkama W.; Mahidol UniversityThe anatomical, histological, and histochemical characteristics of the foregut (FG), midgut (MG), and hindgut (HG), as well as their alterations during the ovarian cycle in female prawns, Macrobrachium rosenbergii, were investigated. The esophagus (ESO), cardia (CD), and pylorus (PY) are the main components of the FG. An epithelium (Ep) with thick cuticle (Cu) layers lining the ESO, and the ESO is encircled by the ESO glands. The CD has a thick musculature, whereas the Ep of the PY are characterized by numerous villi and columnar Ep cells with a thinner layer of Cu. The inner longitudinal (LM) and the outer circular (CM) muscles were both present in the PY. The MG is lined by Ep cells which are connected to the basement membrane, and it lacks Cu. Microvilli, and subapical vacuoles are visible on the apical surface of Ep cells of the MG. The outermost layer is characterized by a dense strip of elastic fibers and a cluster of collagen fibers. The HG has the Ep cells with a thin Cu layer, and the HG glands form a rosette-like structure. The HG is surrounded by the CM and the LM fibers. The reactivities of Periodic Acid Schiff and Alcian Blue in these digestive organs altered throughout the ovarian cycle, and this was supported by the increased expression of mucin levels as ovarian maturation progressed. Our results offer novel and significant insights into the anatomical and histochemical structures of these digestive organs, and demonstrate a significant correlation between ovarian development and feeding in the female prawn, M. rosenbergii.Item Metadata only QM/MM Molecular Modeling Reveals Mechanism Insights into Flavin Peroxide Formation in Bacterial Luciferase(2022-01-24) Lawan N.; Tinikul R.; Surawatanawong P.; Mulholland A.J.; Chaiyen P.; Mahidol UniversityBacterial luciferase (Lux) catalyzes oxidation of reduced flavin mononucleotide (FMN) and aldehyde to form oxidized FMN and carboxylic acid via molecular oxygen with concomitant light generation. The enzyme is useful for various detection applications in biomedical experiments. Upon reacting with oxygen, the reduced FMN generates C4a-peroxy-FMN (FMNH-C4a-OO-) as a reactive intermediate, which is required for light generation. However, the mechanism and control of FMNH-C4a-OO- formation are not clear. This work investigated the reaction of FMNH-C4a-OO- formation in Lux using QM/MM methods. The B3LYP/6-31G*/CHARMM27 calculations indicate that Lux controls the formation of FMNH-C4a-OO- via the conserved His44 residue. The steps in intermediate formation are found to be as follows: (i) H+ reacts with O2 to generate +OOH. (ii) +OOH attacks C4a of FMNH- to generate FMNH-C4a-OOH. (iii) H+ is transferred from FMNH-C4a-OOH to His44 to generate FMNH-C4a-OO- while His44 stabilizes FMNH-C4a-OO- by forming a hydrogen bond to an oxygen atom. This controlling key mechanism for driving the change from FMNH-C4a-OOH to the FMNH-C4a-OO- adduct is confirmed because FMNH-C4a-OO- is more stable than FMNH-C4a-OOH in the luciferase active site.Item Metadata only Reaction mechanism and kinetics of the two-component flavoprotein dimethyl sulfone monooxygenase system: Using hydrogen peroxide for monooxygenation and substrate cleavage(2023-01-01) Mangkalee M.; Oonanant W.; Aonbangkhen C.; Pimviriyakul P.; Tinikul R.; Chaiyen P.; Insin N.; Sucharitakul J.; Mahidol UniversityThe dimethyl sulfone monooxygenase system is a two-component flavoprotein, catalyzing the monooxygenation of dimethyl sulfone (DMSO2) by oxidative cleavage producing methanesulfinate and formaldehyde. The reductase component (DMSR) is a flavoprotein with FMN as a cofactor, catalyzing flavin reduction using NADH. The monooxygenase (DMSMO) uses reduced flavin from the reductase and oxygen for substrate monooxygenation. DMSMO can bind to FMN and FMNH− with a Kd of 17.4 ± 0.9 μm and 4.08 ± 0.8 μm, respectively. The binding of FMN to DMSMO is required prior to binding DMSO2. This also applies to the fast binding of reduced FMN to DMSMO followed by DMSO2. Substituting reduced DMSR with FMNH− demonstrated the same oxidation kinetics, indicating that FMNH− from DMSR was transferred to DMSMO. The oxidation of FMNH−:DMSMO, with and without DMSO2 did not generate any flavin adducts for monooxygenation. Therefore, H2O2 is likely to be the reactive agent to attack the substrate. The H2O2 assay results demonstrated production of H2O2 from the oxidation of FMNH−:DMSMO, whereas H2O2 was not detected in the presence of DMSO2, confirming H2O2 utilization. The rate constant for methanesulfinate formation determined from rapid quenched flow and the rate constant for flavin oxidation were similar, indicating that H2O2 rapidly reacts with DMSO2, with flavin oxidation as the rate-limiting step. This is the first report of the kinetic mechanisms of both components using rapid kinetics and of a method for methanesulfinate detection using LC–MS.Item Metadata only Structure and biochemical characterization of an extradiol 3,4-dihydroxyphenylacetate 2,3-dioxygenase from Acinetobacter baumannii(2023-10-01) Pimviriyakul P.; Buttranon S.; Soithongcharoen S.; Supawatkon C.; Disayabootr K.; Watthaisong P.; Tinikul R.; Jaruwat A.; Chaiyen P.; Chitnumsub P.; Maenpuen S.; Mahidol University3,4-Dihydroxyphenylacetate (DHPA) 2,3-dioxygenase (EC 1.13.11.15) from Acinetobacter baumannii (AbDHPAO) is an enzyme that catalyzes the 2,3-extradiol ring-cleavage of DHPA in the p-hydroxyphenylacetate (HPA) degradation pathway. While the biochemical reactions of various DHPAOs have been reported, only structures of DHPAO from Brevibacterium fuscum and their homologs are available. Here, we report the X-ray structure and biochemical characterization of an Fe2+-specific AbDHPAO that shares 12% sequence identity to the enzyme from B. fuscum. The 1.8 Å X-ray structure of apo-AbDHPAO was determined with four subunits per asymmetric unit, consistent with a homotetrameric structure. Interestingly, the αβ-sandwiched fold of the AbDHPAO subunit is different from the dual β-barrel-like motif of the well-characterized B. fuscum DHPAO structures; instead, it is similar to the structures of non-DHPA extradiol dioxygenases from Comamonas sp. and Sphingomonas paucimobilis. Similarly, these extradiol dioxygenases share the same chemistry owing to a conserved 2-His-1-carboxylate catalytic motif. Structure analysis and molecular docking suggested that the Fe2+ cofactor and substrate binding sites consist of the conserved residues His12, His57, and Glu238 forming a 2-His-1-carboxylate motif ligating to Fe2+ and DHPA bound with Fe2+ in an octahedral coordination. In addition to DHPA, AbDHPAO can also use other 3,4-dihydroxyphenylacetate derivatives with different aliphatic carboxylic acid substituents as substrates, albeit with low reactivity. Altogether, this report provides a better understanding of the structure and biochemical properties of AbDHPAO and its homologs, which is advancing further modification of DHPAO in future applications.Item Metadata only Synthetic microbes and biocatalyst designs in Thailand(2023-01-01) Trisrivirat D.; Tinikul R.; Chaiyen P.; Mahidol UniversityFurthering the development of the field of synthetic biology in Thailand is included in the Thai government's Bio-Circular-Green (BCG) economic policy. The BCG model has increased collaborations between government, academia and private sectors with the specific aim of increasing the value of bioindustries via sustainable approaches. This article provides a critical review of current academic research related to synthetic biology conducted in Thailand during the last decade including genetic manipulation, metabolic engineering, cofactor enhancement to produce valuable chemicals, and analysis of synthetic cells using systems biology. Work was grouped according to a Design-Build-Test-Learn cycle. Technical areas directly supporting development of synthetic biology for BCG in the future such as enzyme catalysis, enzyme engineering and systems biology related to culture conditions are also discussed. Key activities towards development of synthetic biology in Thailand are also discussed.Item Metadata only The effects of prostaglandin E2 on gonadal development and germ cell proliferation, and its presence during the gonadal cycle in the sea cucumber, Holothuria scabra(2022-06-30) Nontunha N.; Tinikul R.; Chaiyamoon A.; Vetkama W.; Thongbuakaew T.; Chaichotranunt S.; Poomtong T.; Sobhon P.; Tinikul Y.; Mahidol UniversityProstaglandin E2 (PGE2) plays important roles in several physiological processes, including reproduction of several marine invertebrate species. The aims of the present study were to characterize the putative prostaglandin E synthase (PGES), an enzyme that is involved in the PGE2 biosynthetic pathway, in the sea cucumber, H. scabra. We then examined the presence and distribution of PGES and PGE2 in gonadal tissues using RT-qPCR and immunohistochemistry, and further investigated the effects of PGE2 on gonadal development and germ cell proliferation by using functional assays. Transcriptomic data and molecular cloning analyses from gonadal tissues of H. scabra were performed, and these tissues were found to have the PGES in H. scabra, namely Hs-cPGES3. By using RT-qPCR, the Hs-cPGES3 mRNA expression levels were higher at the early stages than the late stages of the gonadal cycle. In addition, the presence of PGE2 and its corresponding enzyme, Hs-cPGES3, was detected in the gonads with the highest intensities appearing in gamete cytoplasm during the early stages than those at the late stages. Functionally, PGE2, at the doses of 50 and 500 ng/g BW, significantly promoted gonadal development, gonado-somatic index (GSI), gonadal tube diameters, and increased germ cell proliferation, compared with those of the control animals (P < 0.05). Taken together, our findings are the first to demonstrate the presence of PGE2 and its biosynthetic-associated enzymes in the gonadal tissues, and its effects on gonadal development and germ cell proliferation in this sea cucumber. The knowledge gained would improve understanding of PGE2 mechanism in controlling gonadal development, which could be useful in enhancing the production of seeds for aquaculture of H. scabra.
