Recombinant Photobacterium profundum Oligoribonuclease (orn)

What is Photobacterium profundum Oligoribonuclease (orn)?

Oligoribonuclease (Orn) from Photobacterium profundum is a specialized 3'-5' exoribonuclease that plays a crucial role in the final step of RNA degradation. Unlike other exoribonucleases, Orn exhibits a unique substrate preference for diribonucleotides, effectively serving as a diribonuclease that completes the terminal step of RNA degradation pathways . P. profundum is a Gram-negative bacterium originally collected from the Sulu Sea, with a genome consisting of two chromosomes and an 80 kb plasmid . The Orn enzyme is part of the cellular machinery that ensures proper RNA turnover and recycling of nucleotides, which is essential for cellular viability and function.

How does P. profundum adapt to high pressure environments?

P. profundum is a moderate piezophile that grows optimally at 28 MPa (approximately 280 times atmospheric pressure) and 15°C . The organism has evolved several adaptations to thrive under high hydrostatic pressure conditions. One key adaptation involves the accumulation of specific intracellular solutes. When grown at optimal pressure (20-30 MPa), P. profundum cells preferentially increase intracellular concentrations of beta-hydroxybutyrate (beta-HB) and beta-HB oligomers, while amino acid pools remain relatively constant . These beta-HB molecules represent a novel class of osmolytes termed "piezolytes," whose cellular levels respond to both hydrostatic pressure and osmotic pressure . Additionally, proteome analysis reveals that proteins involved in key metabolic pathways are differentially expressed under varying pressure conditions, with glycolysis/gluconeogenesis pathway proteins up-regulated at high pressure, while several oxidative phosphorylation pathway proteins are up-regulated at atmospheric pressure .

What is the primary function of Oligoribonuclease in RNA degradation?

Oligoribonuclease serves as the terminal enzyme in RNA degradation pathways, specifically hydrolyzing diribonucleotides to mononucleotides. RNA degradation is a step-wise process that begins with endonuclease-catalyzed cleavages, followed by exoribonuclease processing of the resulting fragments . While other exoribonucleases can process longer oligoribonucleotides down to diribonucleotide entities, Orn is uniquely responsible for converting these diribonucleotides to mononucleotides . This function is essential for completing RNA degradation and maintaining cellular nucleotide pools. The importance of this role is underscored by the fact that Orn is required for viability in many γ-proteobacteria, unlike all other known 3'-5' exoribonucleases, indicating that Orn catalyzes a particularly crucial step in RNA turnover .

What methodologies are used to study Orn activity?

Historically, Orn activity has been studied using several experimental approaches. Early investigations utilized 3H polyuridine (poly(U)) substrates incubated with Orn, with products analyzed by paper chromatography, though this offered limited resolution . More recent methods involve incubating Orn with oligoribonucleotides tagged at their 5' terminus with fluorophores, allowing detection of reaction products via denaturing polyacrylamide gel electrophoresis . For structural characterization, X-ray crystallography has been employed to determine substrate-bound Orn structures, revealing an active site optimized for diribonucleotide binding . Proteomics approaches, particularly label-free quantitative proteomics using mass spectrometry, have been instrumental in studying Orn expression levels under different environmental conditions, such as varying hydrostatic pressures . These methodological approaches provide complementary insights into Orn's function, structure, and regulation.

How does Orn's substrate specificity differ from other cellular exoribonucleases?

Orn exhibits exquisite substrate preference for diribonucleotides, distinguishing it from other cellular exoribonucleases that process longer oligoribonucleotides. Crystal structures of substrate-bound Orn reveal an active site specifically optimized for diribonucleotide binding and hydrolysis . This unique substrate specificity explains why Orn cannot be functionally replaced by other exoribonucleases such as RNase R and RNase II, despite these enzymes sharing activity toward longer oligoribonucleotides . The molecular basis for this specificity appears to be structural constraints in the active site that accommodate diribonucleotides preferentially. In the RNA degradation pathway, while enzymes like RNase II and RNase R can processively degrade RNA down to short oligoribonucleotides including diribonucleotides, only Orn can efficiently cleave these diribonucleotides to mononucleotides, completing the degradation process . This specialized role makes Orn essential for cellular viability in many γ-proteobacteria, including P. profundum.

What is the role of Orn in bacterial cyclic-di-GMP signaling pathways?

Orn plays a key role in bacterial cyclic-di-GMP (c-di-GMP) signaling, which controls a wide range of cellular pathways including cell adhesion, biofilm formation, and virulence in response to environmental cues . The degradation of c-di-GMP occurs through a two-step process, with a linear pGpG diribonucleotide intermediate . While phosphodiesterases (PDEs) are responsible for linearizing c-di-GMP to pGpG, Orn has been identified as the primary enzyme that degrades pGpG . This function is particularly important because the accumulation of pGpG in Orn-deficient cells can inhibit upstream phosphodiesterases that degrade c-di-GMP, thereby leading to elevated c-di-GMP levels and triggering associated phenotypes . Through this mechanism, Orn serves as a crucial regulator in c-di-GMP signaling networks, influencing numerous cellular processes controlled by this second messenger.

How does hydrostatic pressure affect gene expression and protein function in P. profundum?

P. profundum exhibits complex gene expression patterns in response to hydrostatic pressure changes. Transcriptomic analyses have identified a group of 22 genes with expression profiles similar to OmpH, an outer membrane protein transcribed in response to high hydrostatic pressure . Proteomic studies reveal that proteins involved in the glycolysis/gluconeogenesis pathway are up-regulated at high pressure (28 MPa), while several proteins involved in oxidative phosphorylation are up-regulated at atmospheric pressure (0.1 MPa) . Nutrient transport systems are also affected by pressure changes. For example, phosphate transport ATP-binding cassette (ABC) system components, including phosphate ABC transporter ATP-binding protein (PBPRA1391) and phosphate ABC transporter periplasmic phosphate-binding protein (PBPRA1394), are down-regulated at 28 MPa compared to 0.1 MPa . This suggests that either phosphate requirements differ at various sea depths or that the transport system has evolved to function more efficiently at high pressure, requiring greater abundance at low pressure to compensate for reduced effectiveness .

What structural adaptations allow P. profundum proteins to function under high pressure?

The deep-sea environment imposes unique constraints on protein structure and function due to high hydrostatic pressure. While specific structural information about P. profundum Orn is not detailed in the search results, general principles of protein adaptation to high pressure in piezophiles can be inferred. Proteins from piezophiles typically exhibit structural features that maintain flexibility and function under compression, often including fewer large hydrophobic cores, increased use of hydrogen bonds and salt bridges, and sometimes fewer cavities that could collapse under pressure. The increased concentrations of beta-hydroxybutyrate (beta-HB) and beta-HB oligomers observed in P. profundum cells under optimal pressure conditions suggest these molecules may act as chemical chaperones that help stabilize protein structures under pressure . Additionally, the differential expression of specific proteins under varying pressure conditions indicates a complex adaptive response that likely involves both structural modifications and altered expression patterns of proteins with pressure-optimized structures .

What are the implications of Orn deficiency on cellular physiology?

Orn deficiency has profound implications for cellular physiology due to the enzyme's essential role in both RNA degradation and c-di-GMP signaling. In RNA degradation pathways, loss of Orn function leads to accumulation of diribonucleotides, which can disrupt nucleotide pool homeostasis and potentially affect various cellular processes dependent on proper nucleotide balance . Furthermore, in c-di-GMP signaling, Orn deficiency results in accumulation of the linear pGpG intermediate, which inhibits upstream phosphodiesterases that degrade c-di-GMP . This inhibition causes elevated c-di-GMP levels, triggering phenotypes associated with high cellular c-di-GMP, such as increased biofilm formation and altered virulence in pathogenic bacteria . The essentiality of Orn for viability in many γ-proteobacteria underscores its critical importance in cellular physiology . The unique role of Orn cannot be complemented by other exoribonucleases, making it a potential target for antimicrobial development in pathogenic bacteria where Orn is essential.

How can recombinant P. profundum Orn be used as a research tool?

Recombinant P. profundum Orn, with its unique diribonuclease activity, offers several applications as a research tool. It can be employed to specifically degrade diribonucleotides in biochemical assays, providing a method to analyze the role of these molecules in various cellular processes. In studies of c-di-GMP signaling, recombinant Orn can be used to modulate pGpG levels, enabling researchers to investigate the effects of this signaling intermediate on downstream processes . Additionally, as a pressure-adapted enzyme from a piezophile, recombinant P. profundum Orn provides an opportunity to study the molecular basis of enzyme adaptation to high pressure. Comparative structural and functional analyses between Orn from P. profundum and mesophilic counterparts can yield insights into pressure adaptation mechanisms. The enzyme could also be valuable for biotechnological applications requiring nucleic acid processing under non-standard conditions, particularly those involving high pressure or low temperature environments.

What expression systems are optimal for producing recombinant P. profundum Orn?

While the search results don't specifically address expression systems for P. profundum Orn, general principles for expressing recombinant proteins from piezophilic organisms can be applied. Heterologous expression of P. profundum proteins typically employs E. coli-based systems with cold-adapted strains that can grow at lower temperatures (15-20°C) to better match the native environment of P. profundum. Expression vectors with inducible promoters (such as T7 or tac) allow controlled expression, while affinity tags (His6, GST, or MBP) facilitate purification. Since P. profundum grows optimally at 28 MPa and 15°C , expression conditions should be optimized to ensure proper folding of the recombinant protein. Codon optimization may be necessary due to potential codon usage differences between P. profundum and the expression host. For functional studies, it's important to consider that the recombinant enzyme may exhibit different kinetic properties at atmospheric pressure compared to its native high-pressure environment, necessitating specialized high-pressure equipment for accurate characterization.

What methods can be used to assess Orn activity under varying pressure conditions?

Evaluating Orn activity under different pressure conditions requires specialized equipment and methodologies. High-pressure bioreactors or pressure chambers compatible with enzymatic assays are essential for mimicking the native deep-sea environment. Activity assays typically involve incubating the enzyme with diribonucleotide substrates under varying pressures, followed by analysis of reaction products. HPLC or mass spectrometry can be used to quantify the conversion of diribonucleotides to mononucleotides . Fluorescently labeled substrates offer another approach, allowing real-time monitoring of activity if the pressure chamber permits optical measurements. For comprehensive characterization, enzyme kinetics (Km, Vmax) should be determined across a range of pressures, from atmospheric (0.1 MPa) to the optimal growth pressure of P. profundum (28 MPa) . Comparative analyses with Orn orthologs from non-piezophilic organisms can provide valuable insights into pressure adaptation mechanisms. Additionally, structural studies using techniques like hydrogen-deuterium exchange mass spectrometry can reveal pressure-induced conformational changes that may affect enzyme activity.

How can mutagenesis studies identify key residues for Orn substrate specificity?

Site-directed mutagenesis represents a powerful approach for identifying critical residues involved in Orn's specificity for diribonucleotides. Based on crystal structures of substrate-bound Orn that reveal an active site optimized for diribonucleotides , targeted mutations can be introduced at residues that contact the substrate or participate in catalysis. Conserved residues in the active site should be prioritized, particularly those that differ between Orn and other exoribonucleases with broader substrate specificity. After generating mutant variants, enzyme activity assays using diribonucleotides and longer oligoribonucleotides can assess changes in substrate preference. Kinetic parameters (Km, kcat) should be determined for each substrate to quantify specificity changes. Structural analyses of mutant proteins through X-ray crystallography or cryo-EM can reveal how mutations alter substrate binding. Additionally, complementation studies in Orn-deficient bacterial strains can evaluate the functional consequences of mutations in vivo, particularly their effects on c-di-GMP signaling and RNA degradation pathways .

Comparison of P. profundum Orn with orthologs from other bacterial species

Expression profile of Orn under varying pressure conditions

While the search results don't provide specific data on Orn expression under different pressure conditions in P. profundum, they do indicate that numerous proteins show differential expression based on hydrostatic pressure . P. profundum exhibits a complex expression pattern with groups of genes responding to pressure changes, including 22 genes with expression profiles similar to OmpH, which is upregulated at high pressure . By inference, Orn expression may be regulated in response to pressure, particularly given its essential role in cellular processes. The pressure-responsive ToxR protein, which regulates genes in a pressure-dependent manner, could potentially influence Orn expression directly or indirectly . Further research employing transcriptomic and proteomic analyses specifically targeting Orn expression across pressure gradients would be valuable for understanding how this essential enzyme is regulated in response to environmental pressure changes.

What are the potential applications of pressure-adapted enzymes from piezophiles?

Pressure-adapted enzymes from piezophiles like P. profundum have significant potential applications in biotechnology, bioremediation, and fundamental research. These enzymes may offer advantages for industrial processes requiring high pressure conditions, such as certain food processing techniques, deep-sea bioremediation of pollutants, or biotransformations under pressure. The unique catalytic properties of piezophilic enzymes could enable novel reactions or improved efficiency under non-standard conditions. For Orn specifically, its diribonuclease activity could be harnessed for applications requiring selective degradation of short RNA species or for manipulating c-di-GMP signaling pathways in bacterial systems . Additionally, understanding the structural basis of pressure adaptation in these enzymes could inform protein engineering efforts aimed at creating pressure-resistant variants of industrial enzymes. The exceptional substrate specificity of Orn also makes it a valuable tool for studying RNA degradation pathways and developing targeted approaches to modulate RNA metabolism in research and therapeutic contexts.

How might Orn be targeted for antimicrobial development?

The essentiality of Orn in many γ-proteobacteria, including several pathogenic species, positions it as a potential target for novel antimicrobial development . Since Orn cannot be functionally replaced by other exoribonucleases, inhibitors specific to Orn would likely have bactericidal effects on organisms where it is essential. A rational drug design approach could leverage the structural information about Orn's active site, which is optimized for diribonucleotides , to develop compounds that selectively bind and inhibit the enzyme. High-throughput screening of compound libraries against recombinant Orn could identify lead molecules for further development. Potential inhibitors might include diribonucleotide analogs that bind but resist hydrolysis, or small molecules that occlude the active site. The unique substrate specificity of Orn provides an opportunity for developing highly specific inhibitors with potentially fewer off-target effects. Additionally, disrupting Orn's role in c-di-GMP signaling could alter bacterial behaviors like biofilm formation and virulence, offering alternative therapeutic strategies beyond direct bactericidal effects .