Biology subjects

Saikrishnan, K.

Publications and source records attributed to Saikrishnan, K..

5 recordsLinked to original sources

bioRxiv2026

Biochemical and kinetic properties of a Type III restriction-modification enzyme Mbo45V from the host-adapted pathogen Mycoplasma bovis

Type III restriction-modification (RM) enzymes are prominent bacterial defense against bacteriophage and invading foreign DNA that also modulate the hosts epigenetic landscape. Genome analysis of the host-adapted Mycoplasma bovis PG45 that has a very small genome revealed a Type III RM locus comprising one res and three mod genes. We characterized Mbo45V, a representative enzyme encoded by this locus. The enzyme forms a heterotrimeric complex consisting of two Mod subunits and one Res subunit. Mbo45V recognizes the asymmetric sequence 5'-YAATC-3' (Y = T/C) and cleaves DNA having at least two head-to-head oriented sites [~]26-28 bp away from the recognition site. Methylation of the second adenine of the target site using cofactor S-adenosylmethionine (SAM) protects DNA from restriction, while the SAM analogue sinefungin enhances DNA binding and cleavage. Kinetic studies reveal that Mbo45V exhibits relatively weak DNA binding affinity and an unusually high Km for SAM, indicating low cofactor affinity compared to prototypical enzymes such as EcoP15I. ATPase activity is strongly stimulated by cognate DNA and is inhibited upon methylation of the substrate, suggesting a regulatory interplay between methylation and restriction functions. Comparative analysis indicates that, although Mbo45V shares core mechanistic features with prototypes from Escherichia coli, its kinetic parameters are distinct. These differences likely reflect adaptation to the stable intracellular environment of M. bovis, in contrast to the fluctuating conditions encountered by the enteric bacteria.

biochemistry↗

bioRxiv2026

Emergence of a complex logic gate from the integration of slippage-induced frameshift mechanisms

Phase variation is viewed as a simple stochastic ON/OFF switch helping bacteria survive unpredictable environments. In minimal-genome pathogens like Mycoplasma bovis, simple sequence repeats (SSRs) introduce frameshifting InDels in key phase-variable genes, such as the Type III restriction-modification mod genes, typically assumed to result in binary expression. This study revisits this assumption using a heterologous E. coli system and single-cell mEGFP-based fluorescence profiling of M. bovis mod1 gene fragment containing SSR to determine if "OFF" states are truly silent. We find that the frameshifted construct remains active, showing low-level, heterogeneous expression in a frame-dependent manner, via frameshift suppression. This creates a range of expression rather than a strict binary switch. These findings suggest that phase-variable SSRs can function with upstream switches to form complex XNOR Boolean logic gates. This demonstrates that sophisticated logic gates can emerge directly from coding sequence architecture enhancing diversity and adaptability to promote evolutionary resilience in compact bacterial genomes.

molecular biology↗

bioRxiv2025

Mechanism of substrate binding by the SARS-CoV-2 NiRAN domain and modulation of its activities during replication

The SARS-CoV-2 Nidovirus RdRp-associated nucleotidyltransferase (NiRAN) domain initiates viral genome capping by RNAylating nsp9 with the 5'-pppA-end of the genome followed by GDP-dependent deRNAylation to form the core capped GpppA-genome. Additionally, it cycles nsp9 through NMPylation-deNMPylation to generate GpppN. It is unclear how the distinct substrates, 5'-pppA-RNA and NTP, are bound, and how NiRAN balances RNAylation versus NMPylation. Earlier models proposed a common base-up pose for both the substrates. Here, structure-guided mutagenesis and reconstitution assays show that 5'-pppA indeed binds base-up during RNAylation, revealing that nsp12-Asp711 confers adenine selectivity, whereas, NTP adopts a perpendicular base-out pose during NMPylation. NiRAN intrinsically favors NMPylation over RNAylation, but nsp13 NTPase activity flips this preference. RNAylation weakens when RdRp is RNA-bound or replicating it, suggesting that a trans-acting NiRAN associated with an RNA-free RdRp performs capping. These findings provide insights into the orchestration of NiRAN activities and potential druggable sites for anti-viral therapeutics.

molecular biology↗

bioRxiv2025

Remodeling of nucleosome by a DNA translocating bacterial restriction-modification enzyme

A eukaryotic cell has specialized ATP-dependent chromatin remodelers, such as SWI/SNF, to unfurl DNA from nucleosome for functional processing. The ATPase that powers the movement of the chromatin remodeler on the DNA (translocation) is evolutionarily related to those powering the translocation of the functionally distinct bacterial restriction-modification (RM) enzymes. The collision of a SWI/SNF chromatin remodeler and a nucleosome, results in sliding/ejection of the constituent histone octamer, while two converging ATP-dependent RM enzymes catalyze DNA cleavage. Here, we investigate if an ATP-dependent Type ISP RM enzyme, an active and directional translocase, can remodel nucleosomes. Our results reveal that in presence of ATP, a Type ISP RM enzyme can displace the octamers from not just mononucleosomes but also two tandem nucleosomes. However, a Type III RM enzyme, which employs a homologous ATPase as a switch to facilitate bidirectional 1D diffusion along the DNA, fails to remodel the nucleosome. This implies that an actively translocating Type ISP RM enzyme generates sufficient force for chromatin remodeling, and may serve as artificial sequence-specific chromatin remodelers.

molecular biology↗

bioRxiv2022

Sulfur-mediated chalcogen versus hydrogen bonds in proteins: a seesaw effect in the conformational space

Divalent sulfur (S) form chalcogen bond (Ch-bond) via its {sigma}-holes and hydrogen bond (H-bond) via its lone-pairs. Relevance of these interactions and their interplay for protein structure and function is unclear. Based on the analyses of the crystal structures of small organic/organometallic molecules and proteins, and their Molecular Electrostatic Surface Potential, we show that the reciprocity of the substituent-dependent strength of the {sigma}-holes and lone-pairs correlate with the formation of either Ch-bond or H-bond. In proteins, disulfide-bonded cystine preferentially forms Ch-bond, metal-chelated cysteine forms H-bond, while methionine forms either of them with comparable frequencies. This has implications to the positioning of these residues and their role in protein structure and function. Computational analyses reveal that the S-mediated interactions stabilize protein secondary structures by mechanisms such as helix capping, protecting free {beta}-sheet edges by negative-design, and augmenting the stability of {beta}-turns. We find that Ch-bond can be as strong as H-bond. The study highlights the importance of S-mediated Ch-bond and H-bond for understanding protein folding and function, development of improved strategies for protein/peptide structure prediction and design, and structure-based drug discovery.

biochemistry↗