Dr. Apurba Kumar Sau

The primary focus of our research is to understand how enzymes/receptors work at the molecular level, which helps achieve an in-depth mechanistic understanding and thereby provides a more effective way to modulate their functions. We are mainly interested in enzymes/receptors that are important targets for the design of antibiotics and other antimicrobial agents. The functional studies, along with the structural data, may provide a basis for designing inhibitors/drug candidates for therapeutic purposes. We are using a combination of molecular biology, biochemical, structural, and computational studies, such as steady-state kinetics, enzyme assays, rapid transient kinetics, mass spectrometry, circular dichroism, fluorescence, isothermal calorimetry, X-ray crystallography, protein oligomeric studies, protein interaction studies, thermal denaturation, and Molecular Dynamics simulations to address these problems.

1. Interferon-induced GTPases

Interferons (IFNs) are immunomodulatory cytokines with antipathogenic and antiproliferative effects. Human guanylate-binding proteins (hGBPs), among the most abundant proteins induced by IFN-gamma, are 65-67 kDa GTPases found in all vertebrates. Of the seven known hGBP homologs, hGBP1 and hGBP2 are highly overexpressed and central to cell-autonomous innate immunity. Uniquely, these hydrolyze GTP sequentially to GDP and then GMP, though product ratios differ: hGBP1 predominantly yields GMP, hGBP2 mainly GDP, and hGBP3 produces GMP as a minor product (30%); hGBP5 produces no GMP, and other homologs remain uncharacterized in this regard.

hGBPs exhibit broad antiviral, antibacterial, and antiproliferative activity. hGBP1 acts against hepatitis C, dengue, influenza A, vesicular stomatitis, encephalomyocarditis, and KSHV, as well as against Shigella flexneri, Chlamydia trachomatis, and Toxoplasma gondii. Recently, GMP formation by hGBP1 has been reported to be important for its antiviral and antibacterial activities. For instance, the protein-catalyzed stimulation of GMP formation is crucial for suppressing hepatitis C virus proliferation. This GMP has also been shown to restrict Chlamydia growth by activating the NLRP3 inflammasome. However, the relationship between GTPase activity and these functions remains unclear. Moreover, no study has explored the mechanism of substrate hydrolysis by this protein. The structure-function relationship is important for understanding how these proteins function at the molecular level.

Mechanistically, we previously identified an alpha helix in hGBP1's intermediate domain acting as an internal GAP (J. Mol. Biol. 2009, 386, 690-703, Biophysical Journal 2010, 99, 2235-2244). More recent work shows that GMP formation in hGBP1 depends on repositioning of the catalytic loop, driven by an H-bond between W79's indole N and K76's main-chain carbonyl, along with W79 solvent exposure (FEBS Journal 2021, 288, 2970-2988). In hGBP2, this H-bond is absent, so the catalytic loop fails to reposition toward GTP's beta-phosphate, yielding less GMP (FEBS Journal, 2019, 286, 4103-4121; Biochemistry 2023, 62, 1509-1526). In hGBP3, the H-bond exists, but the W79 region doesn't undergo the necessary conformational change after gamma-phosphate cleavage, again limiting GMP output (J. Biol. Chem. 2024, 300, 105696-105723). Together, these findings suggest that differences in post-cleavage catalytic loop repositioning are an important determinant of the variation in GMP formation among these homologs. We also showed that the helical domain of hGBP3 is essential for the anti-HCV activity through hexamer formation (Biochemistry, 2024, 63, 2892-2903).

2. Biosynthetic pathways in Helicobacter pylori

Helicobacter pylori, a human pathogen, infects half of the world's population and causes chronic active gastritis, which can lead to peptic ulcer disease, gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. Current antibiotic therapy requires a combination of drugs with long-term treatment. Moreover, the emergence of drug-resistant strains in human pathogens has gained further attention, leading to the development of new drugs. We are therefore interested in the biosynthetic pathways of the microorganism that are not present in mammals and may therefore be attractive targets for the design of new antibiotics. 

H. pylori arginase and N-carbamoyl putrescine amidase (CPA), enzymes of arginine metabolism and polyamine biosynthesis, represent promising targets, as polyamines are vital for bacterial survival. We identified a unique 13-residue motif in H. pylori arginase found only in gastric pathogens, offering a specific site for therapeutic development (Biochemistry, 2013, 52, 508-519; Biophysical Journal, 2017, 112, 1120–1134; Biochemical Journal, 2019, 476, 3595-3614; Biochemical Journal, 2021, 478, 871-894).

In H. pylori, the polyamine putrescine is produced solely via the metabolism of N-carbamoyl putrescine by CPA, making CPA a crucial enzyme; its absence in humans further underscores its potential as a therapeutic target. We discovered a unique tryptophan cluster near the CPA catalytic site and showed its role in oligomerization and the formation of a catalytically competent conformation (Biophysical Journal, 2021, 121, 248-262). We also identified a five-residue motif that forms a short helix essential for octamer formation and, thus, catalysis in H. pylori CPA (Biochemistry, 2025, 64, 3280–3298).

Our findings were highlighted on the front page 

Awards:
. Fellow of the National Academy of Sciences, India (2017)
. National Bioscience Award for Career Development (2013)