Recombinant Nitrosomonas europaea Lysine--tRNA ligase (lysS), partial

What is Nitrosomonas europaea Lysine--tRNA ligase (lysS) and what is its role in protein synthesis?

Nitrosomonas europaea Lysine--tRNA ligase (lysS) is an aminoacyl-tRNA synthetase responsible for attaching lysine to its cognate tRNA during protein synthesis. This enzyme belongs to the broader family of lysyl-tRNA synthetases, which exist in two unrelated forms: Class I (LysRS1) and Class II (LysRS2). These enzymes catalyze the aminoacylation of tRNA^Lys with lysine, a critical step in translation .

The function of lysS is particularly interesting in Nitrosomonas europaea, an ammonia-oxidizing bacterium where certain amino acids, including lysine, have been shown to inhibit growth . This suggests a complex relationship between the organism's metabolism, protein synthesis machinery, and environmental conditions. Understanding lysS function provides insights into N. europaea's protein synthesis regulation under various growth conditions.

How does Nitrosomonas europaea lysS compare structurally to lysyl-tRNA synthetases in other organisms?

While specific structural data for N. europaea lysS is limited in the available literature, comparative analysis can be made based on knowledge of lysyl-tRNA synthetase diversity across species:

Lysyl-tRNA synthetases exist in two structurally distinct forms:

• Class I (LysRS1): Found predominantly in archaea and some bacteria

• Class II (LysRS2): Found in eukarya, most bacteria, and a few archaea

Based on taxonomic patterns, N. europaea lysS likely belongs to the Class II family (LysRS2), though this would require experimental confirmation. Structural differences between the classes are significant, particularly in the lysine-binding site. LysRS1 has a more compact binding site than LysRS2, affecting their respective abilities to accommodate lysine analogues with backbone substitutions .

What experimental approaches are most effective for measuring recombinant N. europaea lysS activity?

For researchers studying N. europaea lysS activity, several methodological approaches can be employed:

Aminoacylation Assays

The gold standard for lysS activity measurement is the aminoacylation assay. Based on techniques used for other lysyl-tRNA synthetases, this can be performed as follows:

• Express and purify the recombinant enzyme (e.g., using intein fusion systems as described for E. coli LysS )

• Prepare in vitro transcripts of tRNA^Lys substrate

• Conduct aminoacylation reactions under optimized conditions (pH, temperature, buffer composition)

• Monitor attachment of lysine to tRNA by:

  • 32P labeling of tRNA using CCA-adding enzyme followed by aminoacylation and visualization

  • Measuring incorporation of radiolabeled lysine into tRNA

Inhibition Studies

Since lysine appears inhibitory to N. europaea growth, inhibition studies can provide insights into lysS function:

• Test the effect of lysine analogues (e.g., thialysine, S-(2-aminoethyl)-L-cysteine) on enzyme activity

• Determine competitive inhibition constants (Ki values)

• Correlate in vitro inhibition with growth effects in vivo

These approaches can help distinguish lysS-specific effects from broader metabolic impacts of lysine on N. europaea.

How does lysine affect Nitrosomonas europaea growth, and what implications does this have for lysS research?

Research has shown that L-lysine exhibits inhibitory effects on Nitrosomonas europaea growth, an unexpected finding given that amino acids often serve as potential carbon or nitrogen sources for bacteria .

In detailed growth studies, L-lysine along with L-histidine, L-threonine, L-valine, L-methionine, and L-arginine demonstrated inhibitory effects on both nitrite formation and protein synthesis in N. europaea cultures. This contrasts with amino acids like L-glutamic acid, L-aspartic acid, L-serine, and L-glutamine, which enhanced growth .

The inhibitory effect of lysine presents several implications for lysS research:

• Potential feedback mechanisms: The inhibition might suggest regulatory feedback between free lysine levels and lysS activity

• Metabolic interference: Lysine might interfere with ammonia oxidation or other critical metabolic processes

• Transport competition: Lysine may compete with ammonia for transport mechanisms

• Experimental design considerations: Researchers must carefully control lysine concentrations in growth media when studying N. europaea lysS

These findings suggest that unlike many organisms where amino acid supplementation enhances growth, N. europaea has evolved distinct metabolic responses to lysine that may relate to its specialized ecological niche and energy metabolism based on ammonia oxidation .

How can N. europaea lysS be potentially used in genetic code expansion and synthetic biology?

The field of genetic code expansion relies on orthogonal aminoacyl-tRNA synthetase/tRNA pairs that do not cross-react with the host's translational machinery. While N. europaea lysS has not been specifically documented for this application in the search results, potential approaches can be outlined based on related research:

Potential Approaches for Using N. europaea lysS in Genetic Code Expansion:

• Orthogonality assessment: Evaluate whether N. europaea lysS/tRNA^Lys pairs are orthogonal in common expression hosts (E. coli, yeast, mammalian cells) by testing if they interact with endogenous tRNAs or are recognized by host synthetases

• Active site engineering: Based on the approaches used for PylRS systems, the binding pocket of N. europaea lysS could be evolved to accept unnatural amino acids through directed evolution approaches:

  • Positive selection for incorporation of the unnatural amino acid

  • Negative selection against natural amino acid incorporation

• Chimera creation: N. europaea lysS domains could be used to create chimeric synthetases, combining its tRNA binding properties with catalytic domains from other synthetases, similar to approaches described for PylRS chimeras

• tRNA engineering: The N. europaea tRNA^Lys could be engineered with alternative anticodons to respond to stop codons or rare codons while maintaining recognition by its cognate synthetase

The unique properties of N. europaea as an ammonia-oxidizing bacterium with specialized metabolism might provide evolutionary adaptations in its lysS that could be beneficial for genetic code expansion in specific applications.

What are the known inhibitors of lysyl-tRNA synthetases and their potential effects on N. europaea lysS?

Inhibitors of lysyl-tRNA synthetases have significant research value for understanding enzyme mechanisms and potential applications in studies of protein synthesis regulation. For N. europaea lysS, researchers should consider the following inhibitor types:

Lysine Analogues

The naturally occurring metabolic intermediate thialysine (S-(2-aminoethyl)-L-cysteine) is a well-documented lysine analogue that can be incorporated into proteins via LysRS2, effectively inhibiting cellular growth . The sensitivity of N. europaea lysS to thialysine would depend on its structural class:

• If N. europaea lysS is a Class II enzyme (LysRS2), it may readily accept thialysine as a substrate

• If it's a Class I enzyme (LysRS1), its more compact binding site might discriminate against thialysine

Experimental Approach to Inhibitor Studies:

• Perform enzyme kinetics with potential inhibitors to determine Ki values

• Use competitive vs. non-competitive analysis to determine binding sites

• Correlate in vitro inhibition with effects on N. europaea growth

• Perform structure-activity relationship studies with modified inhibitors

Understanding inhibitor profiles could provide insights into the specific properties of N. europaea lysS and potentially explain the growth inhibition observed with lysine supplementation .

How does the expression and function of lysS in N. europaea relate to its unique metabolic characteristics?

Nitrosomonas europaea has a specialized metabolism based on ammonia oxidation for energy generation, making it an obligate chemolithoautotroph. This unique metabolism likely influences lysS expression and function:

Metabolic Context for lysS Function:

• Growth inhibition by lysine: The observed inhibitory effect of lysine on N. europaea growth suggests a potential regulatory relationship between amino acid metabolism and ammonia oxidation pathways

• Carbon assimilation: Though N. europaea primarily fixes carbon dioxide autotrophically, the demonstrated uptake of labeled amino acids indicates partial heterotrophic capabilities that may influence lysS regulation

• Energy limitations: Given the relatively low energy yield from ammonia oxidation, N. europaea likely has evolved efficient protein synthesis machinery, potentially affecting lysS expression levels and catalytic efficiency

• Response to environmental conditions: lysS expression might be regulated in response to changing nitrogen availability, potentially coordinating protein synthesis with energy generation

Research into the transcriptional regulation of lysS under different growth conditions could provide insights into how N. europaea integrates protein synthesis with its specialized energy metabolism. Proteomics studies comparing lysS abundance under autotrophic versus mixotrophic conditions would further illuminate these relationships.

What are the evolutionary implications of lysyl-tRNA synthetase diversity in relation to N. europaea?

The evolutionary distribution of the two unrelated forms of lysyl-tRNA synthetase (LysRS1 and LysRS2) provides a fascinating case study in enzyme evolution and horizontal gene transfer. For N. europaea lysS, this has several implications:

Evolutionary Context:

• Rare coexistence: LysRS1 and LysRS2 are almost never found together in a single organism, with organisms generally containing one or the other. The rare exception is in Methanosarcineae, where they function together with specialized tRNA species

• Domain distribution: LysRS2 (Class II) is found in all eukaryotes, most bacteria, and few archaea, while LysRS1 (Class I) is found in most archaea and some bacteria. Determining which class N. europaea possesses would place it in this evolutionary context

• Selective advantage hypothesis: The structural differences between LysRS1 and LysRS2 suggest potential selective advantages under certain conditions, particularly in handling lysine analogues. This may relate to N. europaea's ecological niche and the observed lysine growth inhibition

• Horizontal gene transfer: The scattered distribution of LysRS types suggests horizontal gene transfer events in evolutionary history. Analysis of N. europaea lysS sequence would help determine if it shows evidence of such transfers

Comparative genomic analysis of lysS sequences across diverse nitrifying bacteria could provide insights into how adaptation to ammonia oxidation might have influenced the evolution of this essential component of the translation machinery.

What methodological approaches should be used when working with recombinant N. europaea lysS in experimental settings?

Researchers working with recombinant N. europaea lysS should consider the following methodological approaches to ensure optimal results:

Expression and Purification:

• Expression system selection: Based on approaches for other lysyl-tRNA synthetases, systems like intein fusion proteins have proven successful for purification to electrophoretic homogeneity

• Protein stability considerations: Storage conditions significantly affect enzyme shelf life. Researchers should evaluate buffer composition (pH, salt concentration, reducing agents) and storage temperature to maximize stability

• Activity assessment: Aminoacylation assays using in vitro transcribed tRNA^Lys are essential for confirming functional activity of purified enzyme

Experimental Design:

• Growth medium formulation: Given lysine's inhibitory effect on N. europaea growth, careful control of amino acid composition in media is essential, especially for in vivo studies

• Cell-free extract preparation: For comparative studies with native enzyme, prepare extracts carefully to preserve activity, as demonstrated in studies measuring lysyl-tRNA synthesis-specific activity in bacterial extracts

• Kinetic parameter determination: Establish standard conditions for determining Km, Vmax, and kcat values, allowing comparison with lysyl-tRNA synthetases from other organisms

• Inhibition studies: Design experiments to determine whether N. europaea lysS has evolved specific mechanisms to discriminate against potentially toxic lysine analogues like thialysine

These methodological considerations will help researchers obtain reliable results when working with this specialized enzyme from an environmentally and metabolically unique bacterium.