Biology subjects

Dubey, B. N.

Publications and source records attributed to Dubey, B. N..

6 recordsLinked to original sources

bioRxiv2026

Dimerization of MilM is essential for catalyzing the pyridoxal-5'-phosphate (PLP)-dependent Cγ-hydroxylation of L-arginine during mildiomycin biosynthesis

MilM from the mildiomycin biosynthetic pathway is a PLP-dependent enzyme, previously annotated as an aminotransferase, but has recently been demonstrated as L-arginine oxidase cum C-hydroxylase. Here, we report detailed biochemical, biophysical, structural modeling, and molecular dynamics simulation-based investigations of MilM from Streptoverticillium rimofaciens B-98891 to elucidate the mechanisms of substrate binding, catalysis, and the role of the active site residues involved in these processes. Our experimental findings confirmed that MilM functions as a stable homodimer, requiring the PLP cofactor and molecular oxygen to transform the L-arginine substrate into 5-guanidino-4-hydroxy-2-oxovaleric acid and 5-guanidino-2-oxovaleric acid through the intermediacy of a possible superoxide radical anion species, while generating H2O2 and NH3 as reaction by-products. Our labeling studies also established that the hydroxyl group in the product is obtained from the solvent, water. The structure-based three-dimensional modeling and simulation of MilM coupled with site-directed mutagenesis further confirmed that, in addition to the catalytic residues Lys232 and His31, active site residues from both the protomers are crucial for stabilization of the PLP cofactor (Ser92, Phe116, Asn164, Asp195, Lys240 from chain A and Tyr89 from chain B) and the substrate (Thr14, Glu17, Asn118, and Arg364 from chain A and Thr259, Ser260 from chain B). Moreover, the molecular dynamics simulation uncovered a dimer-mediated alternating lid mechanism in which large-scale, concerted motions of the dimer interface helices reciprocally expose and occlude the two active sites. This see-saw-like dynamics controls the substrate entry and product release through transiently formed tunnels, while preserving a catalytically protective environment, a critical phenomenon previously left unnoticed in similar PLP-dependent oxidases/hydroxylases. Overall, these findings provide new insights into the substrate/cofactor stabilization and the catalytic mechanism of MilM, a recent member of an emerging family of remarkable PLP-dependent oxidases and help us decode a key puzzle in the mildiomycin biosynthetic pathway.

biochemistry↗

bioRxiv2025

Robust antibiotic sensitization of pathogenic Pseudomonas aeruginosa via negative hysteresis in the cell envelope

Antibiotic combination in time and space is a key strategy to combat antimicrobial resistance. The success of such treatment designs requires their robust efficacy across treatment conditions and a pathogens genomic diversity. This study found that an initial treatment with a {beta}-lactam antibiotic causes robust cellular sensitization towards an aminoglycoside antibiotic across the high-risk human pathogen Pseudomonas aeruginosa, including resistant strains. This phenomenon of cellular sensitization, termed negative hysteresis, is modulated by the Cpx envelope stress response system and linked to membrane stress during growth. The increase in efficacy is achieved through a {beta}-lactam induced elevated cellular uptake of the subsequently administered aminoglycoside. Negative hysteresis and the Cpx system are linked in several cases to the expression of synergistic drug interactions, thus enhancing efficacy of antibiotic combinations. Overall, our study identifies the phenomenon of negative hysteresis as a robustly inducible phenotype and thus a unique focus for optimizing antimicrobial therapy.

evolutionary biology↗

bioRxiv2022

High-resolution crystal structure of a metabolic switch protein in a complex with monomeric c-di-GMP reveals a potential mechanism for c-di-GMP dimerization

Bacterial second messengers c-di-GMP and (p)ppGpp have broad functional repertoires ranging from growth and cell cycle control to the regulation of biofilm formation and virulence. The recent identification of SmbA, an effector protein from Caulobacter crescentus that is jointly targeted by both signaling molecules, has opened up studies on how these global bacterial networks interact. C-di-GMP and (p)ppGpp compete for the same SmbA binding site, with a dimer of the former ligand inducing a conformational change of loop 7 leading to downstream signaling. Here, we report a crystal structure of a partial loop 7 deletion mutant, SmbA{Delta}loop in complex with c-di-GMP determined at 1.4 [A] resolution. SmbA{Delta}loop binds monomeric c-di-GMP strengthening the view that loop 7 is required for c-di-GMP dimerization. In the crystal, SmbA{Delta}loop forms a 2-fold symmetric dimer via isologous interactions with the two symmetric halves of c-di-GMP. Structural comparisons of SmbA{Delta}loop with wild-type SmbA in complex with dimeric c-di-GMP or ppGpp support the idea that loop 7 is critical for SmbA function by interacting with downstream partners. These results underscore the flexibility of c-di-GMP in binding to the symmetric interface between protein subunits. It is envisaged that such isologous interactions of c-di-GMP will be observed in hitherto unrecognized targets.

biochemistry↗

bioRxiv2022

Intermediaries in phosphotransfer: structural features discriminating hybrid histidine kinase Rec domains from response regulator homologs

In two-component systems, the information gathered by the histidine kinase (HK) is relayed to the cognate response regulator (RR). Thereby, the phosphoryl group of the autophosphorylated HK is transferred to the receiver domain of the RR to allosterically activate its effector domain. In contrast, multi-step phosphorelays comprise at least one additional Rec domain (Recinter) that is part of the HK and acts as an intermediary for phosphoryl-shuttling. While RR Rec domains have been studied extensively, little is known about Recinter domains and their potentially discriminating features. As a bona-fide Recinter domain, here we have studied the C-terminal Rec domain of the hybrid HK CckA (CckARec) from Caulobacter crescentus by X-ray crystallography and NMR spectroscopy. CckARec exhibits the canonical Rec-fold, though with a degenerated 4 helix, in which all active site residues are pre-arranged for phosphoryl-binding. BeF3- binding does not alter secondary structure nor the oligomeric state, indicating the absence of allosteric changes, the hall mark of RRs. Based on structural modeling and sequence co-variation analysis, we present a detailed picture for the intramolecular association of the CckA DHp/Rec domains and discuss the role of a FATGUY motif, a distinguishing feature of CckARec orthologs.

biochemistry↗

bioRxiv2019

Precise transcription timing by a second-messenger drives a bacterial G1/S cell cycle transition

Bacteria adapt their growth rate to their metabolic status and environmental conditions by modulating the length of their quiescent G1 period. But the molecular mechanisms controlling G1 length and exit from G1 are poorly understood. Here we identify a key role for the second messenger c-di-GMP, and demonstrate that a gradual increase in c-di-GMP concentration determines precise gene expression during G1/S in Caulobacter crescentus. We show that c-di-GMP strongly stimulates the kinase ShkA, activates the TacA transcription factor, and initiates a G1/S-specific transcription program leading to cell morphogenesis and S-phase entry. C-di-GMP activates ShkA by binding to its central pseudo-receiver domain uncovering this wide-spread domain as a novel signal input module of bacterial kinases. Activation of the ShkA-dependent genetic program also causes c-di-GMP to reach peak levels, which triggers S-phase entry and, in parallel, promotes proteolysis of ShkA and TacA. Thus, a gradual increase of c-di-GMP results in a precisely tuned ShkA-TacA activity window enabling G1/S specific gene expression before cells commit to replication initiation. By defining a regulatory mechanism for G1/S control, this study contributes to understanding bacterial growth control at the molecular level.\n\nGRAPHICAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC=\"FIGDIR/small/675330v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (46K):\norg.highwire.dtl.DTLVardef@1cb0c69org.highwire.dtl.DTLVardef@19bc477org.highwire.dtl.DTLVardef@17f8c89org.highwire.dtl.DTLVardef@e3aa6e_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2019

Hybrid histidine kinase activation by cyclic di-GMP- mediated domain liberation

Cytosolic hybrid histidine kinases (HHKs) constitute major signalling nodes that control various biological processes, but their input signals and how these are processed are largely unknown. In Caulobacter crescentus, the HHK ShkA is essential for accurate timing of the G1-S cell cycle transition and is regulated by the corresponding increase in the level of the second messenger c-di-GMP. Here, we use a combination of X-ray crystallography, NMR spectroscopy, functional analyses and kinetic modelling to reveal the regulatory mechanism of ShkA. In the absence of c-di-GMP, ShkA predominantly adopts a compact domain arrangement that is catalytically inactive. C-di-GMP binds to the dedicated pseudo-receiver domain Rec1 thereby liberating the canonical Rec2 domain from its central position where it obstructs the large-scale motions required for catalysis. Thus, c-di-GMP cannot only stabilize domain interactions, but also engage in domain dissociation to allosterically control activity. Enzyme kinetics data are consistent with conformational selection of the ensemble of active domain constellations by the ligand.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC=\"FIGDIR/small/675454v2_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (11K):\norg.highwire.dtl.DTLVardef@8304borg.highwire.dtl.DTLVardef@19a9d61org.highwire.dtl.DTLVardef@1d27000org.highwire.dtl.DTLVardef@b74d75_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗