Browsing by Author "Radhakrishnan, Manohar"

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    Active Site Characterization of a Campylobacter jejuni Nitrate Reductase Variant Provides Insight into the Enzyme Mechanism
    (ACS, 2024) Yang, Jing; Mintmier, Breeanna; KC, Khadanand; Metzger, Mikayla C.; Radhakrishnan, Manohar; McGarry, Jennifer; Wilcoxen, Jarett; Basu, Partha; Kirk, Martin L.; Chemistry and Chemical Biology, School of Science
    Mo K-edge X-ray absorption spectroscopy (XAS) is used to probe the structure of wild-type Campylobacter jejuni nitrate reductase NapA and the C176A variant. The results of extended X-ray absorption fine structure (EXAFS) experiments on wt NapA support an oxidized Mo(VI) hexacoordinate active site coordinated by a single terminal oxo donor, four sulfur atoms from two separate pyranopterin dithiolene ligands, and an additional S atom from a conserved cysteine amino acid residue. We found no evidence of a terminal sulfido ligand in wt NapA. EXAFS analysis shows the C176A active site to be a 6-coordinate structure, and this is supported by EPR studies on C176A and small molecule analogs of Mo(V) enzyme forms. The SCys is replaced by a hydroxide or water ligand in C176A, and we find no evidence of a coordinated sulfhydryl (SH) ligand. Kinetic studies show that this variant has completely lost its catalytic activity toward nitrate. Taken together, the results support a critical role for the conserved C176 in catalysis and an oxygen atom transfer mechanism for the catalytic reduction of nitrate to nitrite that does not employ a terminal sulfido ligand in the catalytic cycle.
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    Exploring the Antibiotic Potential of a Serine Protease from Solanum trilobatum Against Staphylococcus aureus Biofilms
    (MDPI, 2025-05-07) Radhakrishnan, Manohar; Balu, Kanal Elamparithi; Karthik, Lakshminarayanan; Nagampalli, Raghavendra Sashi Krishna; Nadendla, Eswar Kumar; Krishnasamy, Gunasekaran; Biochemistry and Molecular Biology, School of Medicine
    Background: Multi-antibiotic resistance has become an alarming issue in treating bacterial infections in both community and medical environments. Globally, the scientific community has been exploring multi-antibiotic techniques to find new ways to address this challenge. To address this critical challenge and explore alternative antibiotic treatments, we investigated the potential of Solanum trilobatum, an edible and medicinally important herb plant in Ayurvedic medicine. Methods: Our research focused on a 60 kDa serine protease isolated and purified from the leaves of S. trilobatum, which showed evidence of possessing hydrolase activity. In this study, we examined the capability of the purified enzyme to eradicate preformed biofilms of S. aureus in combination with ampicillin. Additionally, we assessed the stability of the enzyme in the presence of metal ions and detergents. Results: Enzyme kinetics revealed a Vmax of 48.63 µM/min and a Km of 14.08 µM, indicating efficient enzymatic activity. Furthermore, the enzyme exhibited maximum activity at physiological pH, suggesting its potential effectiveness under physiological conditions. Conclusions: Our preliminary findings highlight the promising role of this enzyme as a potential agent to combat S. aureus biofilms, especially when used in conjunction with ampicillin, as an alternative antibiotic approach.
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    Leucine-Rich, Potent Anti-Bacterial Protein against Vibrio cholerae, Staphylococcus aureus from Solanum trilobatum Leaves
    (MDPI, 2022-02-09) Radhakrishnan, Manohar; Palayam, Malathy; Altemimi, Ammar B.; Karthik, Lakshminarayanan; Krishnasam, Gunasekaran; Cacciola, Francesco; Govindan, Lakshmanan; Chemistry and Chemical Biology, School of Science
    A 24 kDa leucine-rich protein from ion exchange fractions of Solanum trilobatum, which has anti-bacterial activity against both the Gram-negative Vibrio cholerae and Gram-positive Staphylococcus aureus bacteria has been purified. In this study, mass spectrometry analysis identified the leucine richness and found a luminal binding protein (LBP). Circular dichroism suggests that the protein was predominantly composed of α- helical contents of its secondary structure. Scanning electron microscopy visualized the characteristics and morphological and structural changes in LBP-treated bacterium. Further in vitro studies confirmed that mannose-, trehalose- and raffinose-treated LBP completely inhibited the hemagglutination ability towards rat red blood cells. Altogether, these studies suggest that LBP could bind to sugar moieties which are abundantly distributed on bacterial surface which are essential for maintaining the structural integrity of bacteria. Considering that Solanum triolbatum is a well-known medicinal and edible plant, in order to shed light on its ancient usage in this work, an efficient anti-microbial protein was isolated, characterized and its in vitro functional study against human pathogenic bacteria was evaluated.