10 research outputs found
Structural and Functional Studies on Salmonella typhimurium Propionate Kinase and Photorhabdus luminescens Oxalate Decarboxylase
No full textAcetate and propionate are low molecular mass carbon compounds found abundantly in the soil. Although these compounds have been extensively used as food preservatives because of their ability to inhibit microbial growth, surprisingly, bacteria such as Escherichia coli and Salmonella typhimurium are able to grow on propionate as their sole carbon and energy source. Only in the presence of glucose, acetate and other short chain fatty acids inhibit microbial growth.
Propionate is produced during the β-oxidation of odd-numbered carbon-chain fatty acids, fermentation of carbohydrates, oxidative degradation of branched-chain amino acids such as valine and isoleucine. They are also produced during the catabolism of threonine, methionine, thymine and cholesterol. In Escherichia coli and Salmonella typhimurium, enzymes involved in the degradation of L-serine and L-threonine to acetate and propionate, respectively, are encoded by the anaerobically regulated tdc operon. L-threonine is anaerobically degraded to propionate in four consecutive reaction steps catalyzed by biodegradative threonine deaminase (TdcB), 2-ketobutyrate formate lyase (TdcE), phosphotransacetylase (Pta) and propionate kinase (TdcD). Detailed studies on the structure and function of two of these enzymes have earlier been carried out in our laboratory. However, these studies did not reveal the precise substrate binding site in Salmonella typhimurium TdcD (StTdcD). It was also not possible to provide a satisfactory explanation of the structural basis of substrate specificity. The present studies were therefore aimed at locating the substrate binding site, elucidating the structural basis of substrate specificity and mechanism of catalysis of StTdcD.
Oxalic acid is toxic to almost all organisms and its excessive occurrence leads to a variety of pathological conditions. In humans and other vertebrates, secretion of oxalic acid leads to formation of low soluble calcium oxalate, which precipitates as kidney stones. Formation of kidney stones is aggravated by lack of enzymes that catabolize oxalate. Oxalate oxidase, oxalate decarboxylase and oxalyl-CoA decarboxylase constitute three distinct categories of oxalate degrading enzymes. Photorhabdus luminescens is a Gram-negative, symbiotic bacterium associated with the entomopathogenic nematodes of the family Heterorhabditidae. Novel insecticidal genes from these symbiotic bacteria are now being examined for their potential in generating pest resistant transgenic plants. As part of this project, the three-dimensional X-ray crystal structure of an oxalate oxidase (OXDC) enzyme from Photorhabdus luminescens (PlOXDC) was determined.
The introductory chapter (Chapter I) of the thesis presents the earlier investigations carried out in the laboratory on the structure and function of StTdcD. It also provides a summary of the earlier literature pertaining to propionate metabolism in S. typhimurium. The crystal structure of StTdcD in the apo form as well as in complex with ADP and the non-hydrolysable nucleotide analog AMPPNP were determined by earlier Dr. Simanshu ( Simanshu et al., 2005 , 2008 ). Subsequently, Dr Chittori determined the structures of the enzyme in complex with various other nucleotides ( Chittori et al., 2013 ). These studies along with enzyme assays performed by Chittori revealed that StTdcD possesses broad specificity and it could be activated by various nucleotides and metal ions and catalyzes phosphorylation of both propionate and acetate ( Chittori et al., 2013 ). In spite of these extensive studies, the precise mode of binding of the substrate propionate to StTdcD could not be elucidated. The chapter also presents a summary of the literature on oxalate, its toxic effects and enzymes that degrade oxalate. The importance of structural and functional studies on oxalate degrading enzymes and other enzymes encoded by Photorhabdus luminescens is also briefly discussed.
All the experimental protocols and computational methods applicable for most of the investigations reported in Chapters 4, 5 and 6 are presented in Chapter II. The experimental procedures described include cloning, overexpression, purification, enzymatic assays, crystallization and X-ray diffraction data collection. Computational methods covered include summary of crystallographic theory and details of various programs used during data processing, structure solution, refinement, model building, validation and analysis. The databases that were used in the course of these investigations are also cited.
The experience gained during attempts to determine the structure of StTdcD by single wavelength anomalous dispersion (SAD) method is described in Chapter III. The impetus for this work was the urge to examine the power of SAD technique making use of a newly acquired rotating anode X-ray generator equipped with a chromium anode. As expected, the structure determined by SAD was very close to the earlier determined structure of StTdcD. The structure contained a citrate, which was part of the crystallization cocktail at the active site. This is in contrast with acetate kinase, where it was found that citrate binds at the dimeric interface. The present studies demonstrated that the identification of a plausible regulatory site at the interface of dimeric structure in acetokinases based on the structure of acetate kinase (Chittori et al., 2013) is not valid for propionate kinase.
Extensive efforts carried out to obtain structures of StTdcD and its mutants StTdcD A88V and StTdcD G207A complexed with either the substrate or substrate analogues provided several crystal structures. In most of these structures, the ligand was bound at a position distinct from the substrate binding site. These structures and their analysis are described Chapter IV. Asn206 was transformed from a disallowed region to an allowed region of the Ramachandran map in these structures whenever an anion was bound at the position corresponding to the γ- phosphate of the nucleotide substrate. This structural transformation might enhance the affinity of the enzyme for the substrate. In the structure of StTdcD A88V in complex with AMPPNP, AMPPNP was found to be cleaved to AMP and PNP either due to catalytic activity of the enzyme or due to radiation damage. The released PNP probably reacted with propionate forming propionyl-pyrophosphate. These structures also demonstrate that the nucleotide site readily accommodates the substrate or substrate analogues in the absence of a bound nucleotide.
StTdcD catalyzes the Mg2+ ion dependent inter-conversion of propionate and ATP to propionyl phosphate and ADP. Two distinct catalytic mechanisms have been proposed for the phosphoryl transfer reaction catalyzed by acetokinase family enzymes: 1) direct-in-line transfer mechanism and 2) triple displacement mechanism (Anthony and Spector, 1972; Matte et al., 1998). In both, the configuration of the transferred phosphate undergoes an inversion, which has been experimentally demonstrated. Structural studies carried out with the view of elucidating the catalytic mechanism of StTdcD is described in Chapter V. Fortunately, it was possible to obtain the crystal structures of StTdcD and its mutants with propionate and AMPPNP bound at the active site. The structure supported an associative SN2 type direct in-line transfer mechanism of catalysis. The studies also revealed that Arg236 and His175 are catalytically important residues. As suggested earlier, Ala88 has a major role in specificity determination. However, Ala88 is not the sole determinant of specificity. Active site volume determining residues, Arg86, His118, Asp143 and the segment Pro116-Leu117-His118 are also important for substrate specificity. The catalytic mechanism proposed in this chapter may also be applicable to other acetokinase family members.
The final Chapter VI describes three different crystal structures of PlOXDC. As expected from sequence similarity with B. subtilis and T. maritima OXDCs, PlOXDC polypeptide was found to possess a bicupin structure. However, the functional unit was a trimer in contrast to BsOXDC which functions as a hexamer. The difference is shown to be due to the disorder in the amino terminal segment of PlOXDC. The polypeptide was truncated during purification by a non-specific cleavage at residue Lys26 either by thrombin used for cleaving the covalently attached GST tag or by some other protease. However, in the crystal structure, the amino terminal 90 residues were disordered. The observed trimeric form of PlOXDC may represent its inherent nature or a result of the missing N-terminal residues. There is some controversy in the literature on whether both or only one cupin domain of the protomer is catalytically active. The structures presented in this chapter provided significant information on the mode of ligand binding to PlOXDC. In one of the structures, EDO was bound to both the cupin domains and was involved in similar interactions with protein atoms. This may imply that the substrate binds at both the sites and both cupin domains may have catalytic function.
The thesis ends with a short note on future perspectives. It is clear that substantial work has been carried out on acetokinases. These studies have provided significant understanding of their structure and function. In the future, appropriate site-specific mutations of the substrate specificity determining residues may be made and their effect on enzyme specificity could be studied. Similarly, mutagenesis experiments could be performed to inter-convert acetate, propionate and butyrate kinases. These studies will provide deeper insights on intricacies of enzyme function. In contrast to the work on short chain fatty acid kinases, work on OXDC should be considered preliminary and further biochemical and structural studies are needed to illustrate the catalytic mechanism and examine if the protein is a suitable candidate for generating transgenic crops resistant to insect pests.
The following manuscripts have been published or will be communicated for publication based on the results presented in the thesis
Facilitating programming comprehension for novice learners with multimedia approach: A preliminary investigation
No full textPotential of Coffee Waste in Enzyme Production under Solid-State Fermentation using Penicillium sp
No full textThis Dissertation / Report is the outcome of investigation carried out by the creator(s) / author(s) at the department/division of Central Food Technological Research Institute (CFTRI), Mysore mentioned below in this page
Feminism in Thamizhselvi's Ponnacharam Novel
No full textFeminism, which is the right half of the world, is being suppressed, ruled, and enslaved for various circumstances and reasons. Feminism is the awakening from this slavery, liberated, thinking about their lives and with the intention of progressing in life. The feminist tendency was being talked about very seriously in the 1990s. Having initially fought against the most horrendous oppression, it now exists at many levels of society. At the beginning of the novel, an eight-year-old girl named Ponnacharam is introduced by the author through a goat-grazing scene. Her birth is viewed as an unwanted birth. Ponnacharam was the eighth child born to the Saatthaiya and Kurunthayi couple. Being born as the eighth, the mother does not dare repent and give it up and puts the child at the feet of the Naganar temple priest. Starting from the fact that he gives the child the name 'Ponnacharam' and sends it away, the life in the sheep pen becomes the life of this child. Ponnacharam transforms herself into a person who loves that life very much. She is shown to have taken to shepherding goats at the age of seven and has been doing it with some kind of commitment. She, who drives herds while tending goats, does not sit in one place while she is in trouble. The fact that she liked the work of herding the sheep with her father, grazing the goat with her mother, boiling rice and water in the hamlet, keeping the sheep in the stable, and cleaning the dung of the sheep would reveal Ponnacharam's professional interest. Thus, the nature of the feminist characters in Tamilselvi's novels can be understood
Structures of substrate- and nucleotide-bound propionate kinase from Salmonella typhimurium: substrate specificity and phosphate-transfer mechanism
No full textKinases are ubiquitous enzymes that are pivotal to many biochemical processes. There are contrasting views on the phosphoryl-transfer mechanism in propionate kinase, an enzyme that reversibly transfers a phosphoryl group from propionyl phosphate to ADP in the final step of non-oxidative catabolism of l-threonine to propionate. Here, X-ray crystal structures of propionate- and nucleotide-bound Salmonella typhimurium propionate kinase are reported at 1.8-2.0 Å resolution. Although the mode of nucleotide binding is comparable to those of other members of the ASKHA superfamily, propionate is bound at a distinct site deeper in the hydrophobic pocket defining the active site. The propionate carboxyl is at a distance of ∼5 Å from the γ-phosphate of the nucleotide, supporting a direct in-line transfer mechanism. The phosphoryl-transfer reaction is likely to occur via an associative SN2-like transition state that involves a pentagonal bipyramidal structure with the axial positions occupied by the nucleophile of the substrate and the O atom between the β- and the γ-phosphates, respectively. The proximity of the strictly conserved His175 and Arg236 to the carboxyl group of the propionate and the γ-phosphate of ATP suggests their involvement in catalysis. Moreover, ligand binding does not induce global domain movement as reported in some other members of the ASKHA superfamily. Instead, residues Arg86, Asp143 and Pro116-Leu117-His118 that define the active-site pocket move towards the substrate and expel water molecules from the active site. The role of Ala88, previously proposed to be the residue determining substrate specificity, was examined by determining the crystal structures of the propionate-bound Ala88 mutants A88V and A88G. Kinetic analysis and structural data are consistent with a significant role of Ala88 in substrate-specificity determination. The active-site pocket-defining residues Arg86, Asp143 and the Pro116-Leu117-His118 segment are also likely to contribute to substrate specificity
Mechanistic features of Salmonella typhimurium propionate kinase (TdcD): insights from kinetic and crystallographic studies
No full textShort-chain fatty acids (SCFAs) play a major role in carbon cycle and can be utilized as a source of carbon and energy by bacteria. Salmonella typhimurium propionate kinase (StTdcD) catalyzes reversible transfer of the γ-phosphate of ATP to propionate during l-threonine degradation to propionate. Kinetic analysis revealed that StTdcD possesses broad ligand specificity and could be activated by various SCFAs (propionate > acetate ≈ butyrate), nucleotides (ATP ≈ GTP > CTP ≈ TTP; dATP > dGTP > dCTP) and metal ions (Mg ≈ Mn > Co ). Inhibition of StTdcD by tricarboxylic acid (TCA) cycle intermediates such as citrate, succinate, α-ketoglutarate and malate suggests that the enzyme could be under plausible feedback regulation. Crystal structures of StTdcD bound to PO (phosphate), AMP, ATP, Ap4 (adenosine tetraphosphate), GMP, GDP, GTP, CMP and CTP revealed that binding of nucleotide mainly involves hydrophobic interactions with the base moiety and could account for the broad biochemical specificity observed between the enzyme and nucleotides. Modeling and site-directed mutagenesis studies suggest Ala88 to be an important residue involved in determining the rate of catalysis with SCFA substrates. Molecular dynamics simulations on monomeric and dimeric forms of StTdcD revealed plausible open and closed states, and also suggested role for dimerization in stabilizing segment 235-290 involved in interfacial interactions and ligand binding. Observation of an ethylene glycol molecule bound sufficiently close to the γ-phosphate in StTdcD complexes with triphosphate nucleotides supports direct in-line phosphoryl transfer
CRITICAL REVIEW OF POTENTIAL FOR IMPLEMENTATION OF LEAN IN THE NIGERIAN BUILDING INDUSTRY
No full textABSTRACT The concept of Lean Construction has been observed to improve workflow reliability, planning and control, particularly in developed and emerging countries like USA, UK, Korea and Brazil. It has been a useful means of achieving project duration reduction and improved cost and quality performance. Predominantly, the Last Planner System, process mapping and other collaborative planning tools of lean construction were utilised in these projects and they accounted for the successes of these projects. However, within under developed and a few developing countries the situation is slightly more at variance. Using Nigeria as a case study for example, the general perception at the moment is that the building industry is mainly characterised by poor project definitions, incomplete project designs and waste generation resulting in uncompleted building projects or poorly completed ones. This paper commences with a review of how lean was applied and why it was successful in the countries identified by previous studies. It then progresses to further review what is currently obtainable in the Nigerian building industry. This then led to a process of addressing the applicability of lean tools within the Nigerian building industry. The results from the review reveal the potential for the application of lean tools within the industry. Sequel to this, the author proposes that Last Planner System of lean be implemented via action research within building industry of Nigeria. It is on the basis of this that further research is being proposed to address this concern
Structure of a 14-3-3ε:FOXO3a<sup>pS253</sup> Phosphopeptide Complex Reveals 14-3‑3 Isoform-Specific Binding of Forkhead Box Class O Transcription Factor (FOXO) Phosphoproteins
No full textThe transcriptional
activity of Forkhead Box O3 (FOXO3a) is inactivated
by AKT-mediated phosphorylation on Serine 253 (S253), which enables
FOXO3a binding to 14-3-3. Phosphorylated FOXO3a binding to 14-3-3
facilitates the nuclear exclusion of FOXO3a, causing cancer cell proliferation.
The FOXO3a/14-3-3 interaction has, therefore, emerged as an important
therapeutic target. Here, we report a comprehensive analysis using
fluorescence polarization, isothermal titration calorimetry, small-angle
X-ray scattering, X-ray crystallography, and molecular dynamics simulations
to gain molecular-level insights into the interaction of FOXO3apS253 phosphopeptide with 14-3-3ε. A high-resolution
structure of the fluorophore-labeled FOXO3apS253:14-3-3ε
complex revealed a distinct mode of interaction compared to other
14-3-3 phosphopeptide complexes. FOXO3apS253 phosphopeptide
showed significant structural difference in the positions of the -3
and -4 Arg residues relative to pSer, compared to that of a similar
phosphopeptide, FOXO1pS256 bound to 14-3-3σ. Moreover,
molecular dynamics studies show that the significant structural changes
and molecular interactions noticed in the crystal structure of FOXO3apS253:14-3-3ε are preserved over the course of the simulation.
Thus, this study reveals structural differences between the binding
to 14-3-3 isoforms of FOXO1pS256 versus FOXO3apS253, providing a framework for the rational design of isoform-specific
FOXO/14-3-3 protein–protein interaction inhibitors for therapy
Factors associated with effects of COVID-19 vaccine among adults in Malaysia
No full textIntroduction: Globally, the needs for more focused research to understand the effects of COVID-19 vaccines among the population to verify the previous research and generate more reliable data. Therefore, this cross-sectional study was aimed to identify the factors associated with the effects of COVID-19 vaccination among adults in Malaysia. Methods: Malaysian people aged 18 and up were enrolled in a cross-sectional online survey. For data collection, a Google form was used to design an online questionnaire which was distributed through social media and in person by the author. Results: A total of 446 responses were analyzed. 58% participants received Pfizer BioNTech Covid 19 vaccine followed by Sinovac (21%) and AstraZeneca (16%). The most reported effect was local reaction pain, swelling and redness at the site of injection with 54.3% of participants. It was seen more in Pfizer BioNTech (56.8%) and AstraZeneca (57.1%) recipients in comparison to Sinovac recipients (42.9%), but this difference was not significant (x2=3.453; p-value=0.315). Tiredness/fatigue was the next most reported effect 51.3% by participants. The other more frequently reported effects were body pain (43.3%), muscle pain (41%), fever (41%) and headache (33%) respectively, which are significantly associated with the type of vaccine. 
Factors Associated with Effects of COVID-19 Vaccine Among Adults in Malaysia
No full textIntroduction: Globally, the needs for more focused research to understand the effects of COVID-19 vaccines among the population to verify the previous research and generate more reliable data. Therefore, this cross-sectional study was aimed to identify the factors associated with the effects of COVID-19 vaccination among adults in Malaysia. Methods: Malaysian people aged 18 and up were enrolled in a cross-sectional online survey. For data collection, a Google form was used to design an online questionnaire which was distributed through social media and in person by the author. Results: A total of 446 responses were analyzed. 58% participants received Pfizer BioNTech Covid 19 vaccine followed by Sinovac (21%) and AstraZeneca (16%). The most reported effect was local reaction pain, swelling and redness at the site of injection with 54.3% of participants. It was seen more in Pfizer BioNTech (56.8%) and AstraZeneca (57.1%) recipients in comparison to Sinovac recipients (42.9%), but this difference was not significant (x2=3.453; p-value=0.315). Tiredness/fatigue was the next most reported effect 51.3% by participants. The other more frequently reported effects were body pain (43.3%), muscle pain (41%), fever (41%) and headache (33%) respectively, which are significantly associated with the type of vaccine. 
