Functional and structural properties of pyridoxal reductase (PdxI) from Escherichia coli. A pivotal enzyme in the vitamin B6 salvage pathway

Pyridoxine 4-dehydrogenase (PdxI), a NADPH-dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5'-phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory-order ternary-complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo-keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B6 in cell extracts of E. coli wild-type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function

Peptidomimetic and Non- Peptidomimetic Derivatives as Possible SARS-CoV-2 Main Protease (Mpro) Inhibitors

To design novel inhibitors of the SARS-CoV-2 main protease (Mpro), we investigated the binding mode of the recently reported α-ketoamide inhibitors of this enzyme. Following, we utilized in-silico screening to identify 168 peptidomimetic and non-peptidomimetic compounds that are high probability Mpro binding candidates. The compounds were synthesized in 5 to 10 mg for initial screening for their potential inhibition of Mpro using Fluorescence Resonance Energy Transfer (FRET) assay. The study was conducted using the main protease, MBP-tagged (SARS-CoV-2) Assay Kit (BPS Bioscience, #79955-2), and the fluorescence due to enzymatic cleavage of substrate measured using BMG LABTECH CLARIOstar™, a fluorescent microplate reader, with an excited/emission wavelength of 360 nm/460 nm, respectively. The FRET assay showed 29 compounds to exhibit lower fluorescence compared to the positive control, indicating inhibitory activity, with three of the compounds exhibiting over 50% enzymatic inhibition. The assay average scores were plotted as dose inhibition curves using variable parameter nonlinear regression to calculate the IC50 values. To design more potent inhibitors, an in-silico molecular docking simulation using the SARS-CoV-2 Mpro crystal structure was conducted to investigate on a molecular level the key binding residues at the active site, as well as the possible binding modes and affinity of the lead inhibitors. Additionally, an in-silico study of the compounds\u27 molecular properties and physicochemical profiles was performed to predict their pharmacokinetic properties and assess their suitability as potential orally active drug candidates.https://scholarscompass.vcu.edu/gradposters/1139/thumbnail.jp

Characterization of the Escherichia coli pyridoxal 5'-phosphate homeostasis protein (YggS): Role of lysine residues in PLP binding and protein stability

The pyridoxal 5'-phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP-dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP-dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function

X-RAY CRYSTALLOGRAPHY: A TOOL FOR UNDERSTANDING PROTEIN FUNCTION AND DRUG DISCOVERY

Structural biology has become an indispensable tool for determining the 3D structures of proteins for a comprehensive understanding of their functions on molecular level, and for elucidating binding cavities that can be targeted for structure-based drug discovery. X-ray crystallography, the most widely used structural biology technique was utilized in addition to other techniques to: (1) understand the role of pyridoxal reductase (PDXI) in pyridoxal 5’-phosphate (PLP) homeostasis; (2) Determine whether the putative PLP binding protein, YggS, is involved in PLP homeostasis; (3) Elucidate the molecular basis underlying Agrobacterium receptor VirA pathogenesis; and (4) Elucidate the structural basis of aromatic aldehydes antisickling activities. PLP is the biologically active form of vitamin B6, serving as a cofactor for over 180 B6 enzymes that are involved in critical biochemical reactions, e.g. amino acid, and neurotransmitter biosynthesis. In prokaryotes, yeasts and plants, PLP is obtained from both de novo and salvage pathways, while only the salvage pathway occurs in humans. Traditionally, the salvage pathway has been described to involve the enzymes, pyridoxal kinase (PLK), pyridoxine 5’-phosphate oxidase (PNPO) and phosphatases that synthesize PLP. Free PLP is regulated by phosphatases, PLK and PNPO. PDXI, with a reductase activity is also known to play an important role in PLP regulation however, only limited information on PDXI structure and function is available. The research objective is to characterize E. coli PDXI with respect to its catalytic conversion of PL to PN, substrate binding specificity, and regulation. PDXI was produced and the crystal structures of the unliganded PDXI, binary PDXI-NADPH, and ternary PDXI-NADPH-PL complexes determined, the first of such structure in the PDXI family of proteins. Biophysical binding studies using isothermal titration calorimetry (ITC) showPDXI binds to NADPH (3.0±0.3 µM) or NADP+ (27.2±1.3 µM) but not B6 vitamers, however, in the presence of NADPH or NADP+, PL or PN binds PDXI, resulting in a KD of 0.3±0.04 µM and 31±0.2 µM, respectively. These observations are consistent with the crystallographic result that showed that PL requires the presence of NADPH to bind PDXI. Kinetic studies show that in the presence of PL and NADPH, the reaction reached fast equilibrium resulting in 96 ± 1.7 % of PL being converted to PN. The crystal structure shows the consensus AKR active site catalytic residue His126 causes steric interaction with PL, explaining why His is replaced by Arg126 in PDXI. Using crystallography in conjunction with other techniques, we provided insight into PDXI catalytic mechanism and substrate specificity. E. coli YggS is a PLP-binding orphan protein, linked to PLP homeostasis. This project tests the hypotheses that YggS functions to scavenge free PLP from the cell and shuttle it apo-B6 enzymes. The crystal structures of wt YggS and several Lys-to-Ala YggS variants, with and without PLP or PNP have been solved. PLP binds covalently to the active site residue K36. Kinetic studies show that only 15% of the K36 bound PLP was transferred from YggS to apo-B6 enzyme, and at a very slow rate (0.14 ± 0.02 min-1). PLP binds YggS with a very high affinity of ~1 nM, but dissociates from YggS very slowly. These observations are inconsistent with YggS serving as a role for B6 activation, and probably not even that of PLP reservoir in the cell. Agrobacterium tumefaciens is a plant pathogen that causes billions of dollars in plant damage. A. tumefaciens uses a transmembrane histidine kinase (VirA) to initiate pathogenesis by enabling VirA to respond to signal upon plant wounding. The research objective is to define the VirA linker/phenol interaction that would help elucidate the molecular basis underlying A. tumefaciens pathogenesis. We have produced soluble protein of the VirA linker region at a purity of ~80% and yield of 10mg/mL for crystallization. Sickle cell disease is the most common inherited hematologic disorder, affecting about 100,000 people in the US, and over 20 million worldwide. A promising therapy is to increase the non-polymer forming oxygenated HbS concentration with aromatic aldehydes. Several aromatic aldehydes alone and/or derivatized with NO-releasing moieties have been studied for their potential treatment of SCD by Dr. Safo’s group. In this study, we used X-ray crystallography to determine the binding modes of aromatic aldehydes with Hb. The crystallographic studies show the compounds to interact with Hb at the α-cleft of Hb, leading to stabilization of the non-polymer forming R-state HbS, explaining the biological activities of the compounds

Design, Synthesis, and Antisickling Investigation of a Nitric Oxide-Releasing Prodrug of 5HMF for the Treatment of Sickle Cell Disease

5-hydroxyfurfural (5HMF), an allosteric effector of hemoglobin (Hb) with an ability to increase Hb affinity for oxygen has been studied extensively for its antisickling effect in vitro and in vivo, and in humans for the treatment of sickle cell disease (SCD). One of the downstream pathophysiologies of SCD is nitric oxide (NO) deficiency, therefore increasing NO (bio)availability is known to mitigate the severity of SCD symptoms. We report the synthesis of an NO-releasing prodrug of 5HMF (5HMF-NO), which in vivo, is expected to be bio-transformed into 5HMF and NO, with concomitant therapeutic activities. In vitro studies showed that when incubated with whole blood, 5HMF-NO releases NO, as anticipated. When incubated with sickle blood, 5HMF-NO formed Schiff base adduct with Hb, increased Hb affinity for oxygen, and prevented hypoxia-induced erythrocyte sickling, which at 1 mM concentration were 16%, 10% and 27%, respectively, compared to 21%, 18% and 21% for 5HMF. Crystal structures of 5HMF-NO with Hb showed 5HMF-NO bound to unliganded (deoxygenated) Hb, while the hydrolyzed product, 5HMF bound to liganded (carbonmonoxy-ligated) Hb. Our findings from this proof-of-concept study suggest that the incorporation of NO donor group to 5HMF and analogous molecules could be a novel beneficial strategy to treat SCD and warrants further detailed in vivo studies

Design, Synthesis, and Investigation of Novel Nitric Oxide (NO)-Releasing Aromatic Aldehydes as Drug Candidates for the Treatment of Sickle Cell Disease

Sickle cell disease (SCD) is caused by a single-point mutation, and the ensuing deoxygenation-induced polymerization of sickle hemoglobin (HbS), and reduction in bioavailability of vascular nitric oxide (NO), contribute to the pathogenesis of the disease. In a proof-of-concept study, we successfully incorporated nitrate ester groups onto two previously studied potent antisickling aromatic aldehydes, TD7 and VZHE039, to form TD7-NO and VZHE039-NO hybrids, respectively. These compounds are stable in buffer but demonstrated the expected release of NO in whole blood in vitro and in mice. The more promising VZHE039-NO retained the functional and antisickling activities of the parent VZHE039 molecule. Moreover, VZHE039-NO, unlike VZHE039, significantly attenuated RBC adhesion to laminin, suggesting this compound has potential in vivo RBC anti-adhesion properties relevant to vaso-occlusive events. Crystallographic studies show that, as with VZHE039, VZHE039-NO also binds to liganded Hb to make similar protein interactions. The knowledge gained during these investigations provides a unique opportunity to generate a superior candidate drug in SCD with enhanced benefits

Unravelling the Perspectives of Day and Night Traders in Selected Markets within a Sub-Saharan African City with a Malaria Knowledge, Attitude and Practice Survey

Background: Malaria is still endemic in sub-Saharan Africa, with a high disease burden. Misconceptions about malaria contribute to poor attitudes and practices, further increasing the burden in endemic countries. Studies have examined the knowledge, attitudes, and practices (KAP) of malaria among different populations. However, there seems to be no available literature reporting on the perspectives of day and night market traders. To the best of our knowledge, this is the first report on malaria KAP with a focus on day and night market traders. Methods: A descriptive cross-sectional study involving day and night market traders in 10 selected markets within the Greater Accra Region of Ghana was carried out. Data were collected from consenting respondents using a structured questionnaire. Results: Of the 760 respondents (33.3% (n = 253) night and 66.7% (n = 507) day traders) interviewed, there was no significant difference between the day and night market traders in terms of malaria KAP. Although the market traders had an overall moderate knowledge (54.0% of the day traders and 56.5% of the night traders), misconceptions about malaria (especially that it could be caused by exposure to the sun) still existed among the traders. Moreover, the majority of the traders who demonstrated high knowledge (43.98%, n = 250) did not always take laboratory tests to confirm their suspicion, indicating poor attitude. Furthermore, the market traders’ choice of drug for malaria treatment (p = 0.001) and preferred malaria treatment type (orthodox or herbal) (p = 0.005) were significantly associated with their knowledge level. Conclusions: Despite the observation that no significant difference in KAP exists between day and night market traders, appropriate health education programs and interventions still need to be directed at misconceptions, poor attitudes, and poor practices revealed by this study. This will ultimately help in the prevention and control of malaria in Ghana, and globally