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Interferon-gamma-inducible human large GTPases, hGBP1 and hGBP2, have a distinctive feature of hydrolyzing GTP to GDP and GMP through successive phosphate cleavages. In hGBP1, GMP is the major product, which is essential for its anti-pathogenic activities. However, its close homologue hGBP2 produces significantly less GMP, despite having a similar active site architecture. The molecular basis for less GMP formation and catalytic residue(s) in hGBP2 are not fully explored. To address these issues, we performed systematic biochemical, biophysical, and microsecond simulation studies. Our data suggest that the less GMP formation in hGBP2 is due to the lack of H-bond formation between the W79 side-chain (located near the active site) and main-chain carbonyl of K76 (present in the catalytic loop) in the substrate-bound hGBP2. The absence of this H-bond could not redirect the catalytic loop toward the beta phosphate after the cleavage of gamma-phosphate, a step essential for enhanced GMP formation. Furthermore, based on the mutational and structural analyses, this study for the first time indicates that the same residue, T75, mediates both phosphate cleavages in hGBP2 and hGBP1. This suggests the conservation of the catalytic residue in hGBP homologues. These findings emphasize the indispensable role of correct catalytic loop repositioning for efficient beta phosphate cleavage. This led us to propose a new substrate hydrolysis mechanism by hGBP1 and hGBP2, which may also be helpful to understand the GTP hydrolysis in other hGBP homologues. Overall, the study could provide insight into how these two close homologues play crucial roles in host-mediated immunity through different mechanisms.

The biodegradation of furfuryl alcohol (FA) in shake flask experiments using a pure culture of Pseudomonas putida (MTCC 1194) and Pseudomonas aeruginosa (MTCC 1034) was studied at 30 °C and pH 7.0. Experiments were performed at different FA concentrations ranging from 50 to 500 mg/l. Before carrying out the biodegradation studies, the bacterial strains were acclimatized to the concentration of 500 mg/l of FA by gradually raising 100 mg/l of FA in each step. The well acclimatized culture of P. putida and P. aeruginosa degraded about 80 and 66% of 50 mg/l FA, respectively. At higher concentration of FA, the percentage of FA degradation decreased. The purpose of this study was to determine the kinetics of biodegradation of FA by measuring biomass growth rates and concentration of FA as a function of time. Substrate inhibition was calculated from experimental growth parameters using the Haldane equation. Data for P. putida were determined as µ = 0.23 h, K = 23.93 mg/l and K = 217.1 mg/l and for P. aeruginosa were determined as µ = 0.13 h, K = 21.3 mg/l and K = 284.9 mg/l. The experimental data were fitted in Haldane, Aiba and Edwards inhibition models.