Recombinant Methanococcus maripaludis PyrE-like protein (MMP0079)

What is MMP0079 and what is its primary function?

MMP0079 is an orotate phosphoribosyltransferase-like protein encoded in the genome of Methanococcus maripaludis S2, a genetically tractable, mesophilic, hydrogenotrophic methanogen. This protein functions primarily in the pyrimidine biosynthesis pathway, catalyzing the conversion of orotate to orotidine 5'-monophosphate (OMP), which is a critical step in the de novo synthesis of UMP and subsequently all pyrimidine nucleotides. The gene is located within the single circular chromosome of M. maripaludis, which contains a total of 1,722 protein-coding genes within its 1,661,137 bp genome . Functionally, MMP0079 shows enrichment for orotate phosphoribosyltransferase activity and is annotated as participating in nucleoside metabolic processes and pyrimidine metabolism .

How does MMP0079 relate to other genes in M. maripaludis?

MMP0079 exists within a complex genetic neighborhood, having a total of 44 gene neighbors in modules 12 and 111 of the M. maripaludis genome . Interestingly, M. maripaludis contains two ORFs encoding orotate phosphoribosyltransferase: MMP0079 and MMP1492 . This duplication is observed in some other archaeal genomes as well, suggesting potential functional redundancy or specialization. Among its neighboring genes are those involved in replication processes, energy metabolism, and several hypothetical proteins, indicating potential co-regulation or functional relationships. The gene immediately adjacent to MMP0079 is MMP0078, which encodes a hypothetical protein whose function remains uncharacterized .

What are the structural characteristics of the MMP0079 protein?

The MMP0079 protein contains characteristic domains expected in orotate phosphoribosyltransferase enzymes, including substrate binding sites for both orotate and phosphoribosyl pyrophosphate (PRPP). Based on motif analysis, four distinct sequence motifs have been predicted in MMP0079, with the following consensus sequences and e-values:

These motifs likely correspond to important functional elements such as active site residues, substrate binding domains, or regions involved in protein-protein interactions . While the high e-values suggest caution in interpretation, these motifs provide valuable starting points for structure-function relationship studies.

What pathways and biological processes involve MMP0079?

MMP0079 is involved in several key metabolic pathways and biological processes within M. maripaludis. Based on functional annotation from multiple systems, MMP0079 participates in:

The primary function of MMP0079 is orotate phosphoribosyltransferase activity, placing it at a critical junction in pyrimidine biosynthesis . In the context of archaeal metabolism, this enzyme enables the organism to synthesize pyrimidine nucleotides necessary for DNA and RNA synthesis. The pyrimidine pathway in M. maripaludis involves the conversion of UTP to CTP (catalyzed by PyrG; Mmp0893) and subsequently to CDP, with further steps leading to the synthesis of all pyrimidine deoxynucleotides required for DNA replication .

What are effective approaches for expressing recombinant MMP0079?

For researchers seeking to express recombinant MMP0079, several methodological considerations are crucial. As an archaeal protein, MMP0079 may present challenges when expressed in bacterial systems due to differences in codon usage, post-translational modifications, and protein folding machinery.

The recommended approach involves:

• Expression system selection: For initial attempts, E. coli BL21(DE3) with codon optimization for archaeal genes is advisable. Alternative expression hosts include Saccharomyces cerevisiae or archaeal expression systems such as Thermococcus kodakarensis if available.

• Vector design: Incorporate a 6×His tag or other affinity tag to facilitate purification. Consider using vectors with archaeal promoters (like hmtB promoter) if expressing in archaeal hosts.

• Expression conditions: For E. coli expression, initial induction with 0.1-0.5 mM IPTG at lower temperatures (18-25°C) may improve solubility. For archaeal enzymes, longer expression periods (overnight) often yield better results than high-concentration, short-term expression.

• Protein solubility enhancement: Co-expression with archaeal chaperones or fusion with solubility-enhancing partners (MBP, SUMO, thioredoxin) can increase soluble yield.

• Purification strategy: Implement a two-step purification process using affinity chromatography followed by size exclusion chromatography to obtain highly pure protein.

Since M. maripaludis is a mesophilic methanogen, its proteins are likely more amenable to expression in mesophilic hosts compared to proteins from hyperthermophilic archaea, which often present greater solubility challenges .

How do the kinetic properties of MMP0079 compare to other PyrE proteins?

While the search results don't provide specific kinetic data for MMP0079, a methodological approach to determining and comparing these properties would include:

• Enzyme activity assay design: The standard assay for orotate phosphoribosyltransferase involves monitoring the conversion of orotate to OMP spectrophotometrically at 295-300 nm. For MMP0079, researchers should establish optimal buffer conditions (likely pH 7.0-8.0) with appropriate metal cofactors (typically Mg²⁺).

• Kinetic parameter determination:

  • Determine Km values for both substrates (orotate and PRPP)

  • Calculate Vmax and kcat

  • Examine the effect of product inhibition

  • Assess pH and temperature optima

• Comparative analysis framework: Compare kinetic properties of MMP0079 with:

  • Its paralog MMP1492 from the same organism

  • PyrE proteins from other methanogens

  • Bacterial and eukaryotic orotate phosphoribosyltransferases

Given that M. maripaludis is mesophilic, MMP0079 likely exhibits optimal activity at moderate temperatures (30-40°C), distinguishing it from thermophilic archaeal homologs. Additionally, researchers should investigate whether MMP0079 displays any allosteric regulation or requires specific ionic conditions reflecting the native cellular environment of M. maripaludis .

What is the functional relationship between MMP0079 and MMP1492?

The presence of two orotate phosphoribosyltransferase genes (MMP0079 and MMP1492) in the M. maripaludis genome presents an intriguing area for research . To investigate their functional relationship, researchers should consider:

• Expression pattern analysis: Using RT-qPCR and/or proteomics to determine whether these paralogs are:

  • Constitutively expressed or differentially regulated

  • Responsive to different environmental conditions

  • Expressed at different levels under normal growth conditions

• Single and double knockout studies: If neither gene is individually essential, creating knockout strains would reveal:

  • Whether they have redundant functions

  • If each has specialized roles under specific conditions

  • Whether the double knockout is lethal (indicating functional redundancy)

• Biochemical characterization: Comparing purified recombinant proteins to determine:

  • Substrate specificity and affinity differences

  • Enzyme kinetics parameters

  • Stability under various conditions

  • Response to potential inhibitors

• Protein localization: Using tagged versions of each protein to determine if they localize to different cellular compartments, potentially indicating specialized roles.

• Interaction partners: Performing pull-down assays to identify whether these paralogs interact with different protein partners.

This study would provide important insights not only into M. maripaludis metabolism but also into the evolution of gene duplication and specialization in archaea .

What regulatory mechanisms control MMP0079 expression?

Understanding the regulation of MMP0079 requires investigation at multiple levels:

• Transcriptional regulation: Analysis of the promoter region reveals potential binding sites for regulatory proteins. The motif information provided in the search results indicates several sequence motifs (IDs 685, 686, 879, and 880) that could represent transcription factor binding sites . To characterize these:

  • Perform chromatin immunoprecipitation experiments to identify proteins binding to the MMP0079 promoter

  • Conduct reporter gene assays with serial deletions of the promoter region

  • Analyze expression under various nutrient conditions and stress states

• Genomic context analysis: MMP0079 has 44 gene neighbors in modules 12 and 111, suggesting potential co-regulation with these genes . Research should examine:

  • Whether these genes form operons

  • If they share common regulatory elements

  • Whether they respond similarly to environmental changes

• Post-transcriptional regulation: Investigate:

  • mRNA stability and half-life

  • Presence of regulatory RNA elements

  • Potential RNA-binding proteins affecting translation

• Metabolic regulation: Examine how pyrimidine levels affect MMP0079 expression:

  • Culture M. maripaludis under varying nucleotide availability conditions

  • Test for feedback inhibition by pathway products

  • Determine if MMP0079 expression responds to growth phase or nutrient limitation

What methods are most effective for studying protein-protein interactions involving MMP0079?

To investigate protein-protein interactions involving MMP0079 in the pyrimidine metabolism pathway, researchers should consider a multi-faceted approach:

• Affinity purification coupled with mass spectrometry (AP-MS):

  • Create a tagged version of MMP0079 (N or C-terminal tag)

  • Express in native M. maripaludis or heterologous system

  • Perform pull-down experiments under varying conditions

  • Identify interacting partners by mass spectrometry

  • Verify interactions with reciprocal pull-downs

• Yeast two-hybrid (Y2H) or bacterial two-hybrid screening:

  • Use MMP0079 as bait to screen against a genomic library of M. maripaludis

  • Focus on candidates from related metabolic pathways

  • Validate positive interactions with alternative methods

• Proximity-based labeling approaches:

  • Fuse MMP0079 with a proximity labeling enzyme (BioID or APEX)

  • Identify proteins in close proximity in vivo

  • Compare interactome under different metabolic states

• Co-immunoprecipitation studies:

  • Develop antibodies against MMP0079

  • Perform co-IP experiments from cell lysates

  • Identify interacting partners by Western blot or MS

• Structural biology approaches:

  • Perform co-crystallization trials with suspected interaction partners

  • Use NMR to detect binding interfaces

  • Employ hydrogen-deuterium exchange mass spectrometry to map interaction surfaces

Given MMP0079's involvement in pyrimidine metabolism, likely interaction partners include other enzymes in this pathway, as well as proteins identified as neighboring genes in modules 12 and 111 . Particular attention should be paid to potential interactions with its paralog MMP1492, regulatory proteins, and enzymes catalyzing adjacent steps in nucleotide biosynthesis.

How can computational approaches enhance our understanding of MMP0079?

Computational methods provide valuable tools for studying MMP0079 structure, function, and evolution:

• Homology modeling and structural prediction:

  • Generate a 3D model using crystal structures of homologous orotate phosphoribosyltransferases as templates

  • Predict active site residues and substrate binding pockets

  • Model how PRPP and orotate bind in the active site

  • Use AlphaFold2 or RoseTTAFold for more accurate structure prediction

• Molecular dynamics simulations:

  • Simulate protein dynamics in different conditions (temperature, pH, salt concentration)

  • Examine conformational changes upon substrate binding

  • Investigate the effects of mutations on protein stability and function

• Phylogenetic analysis:

  • Compare MMP0079 with homologs across archaea, bacteria, and eukarya

  • Trace the evolutionary history of this enzyme family

  • Identify conserved residues as indicators of functional importance

  • Analyze the relationship between MMP0079 and MMP1492 to determine when gene duplication occurred

• Genomic context and co-evolution analysis:

  • Examine gene neighborhood conservation across related species

  • Identify co-evolving residues as indicators of functional interfaces

  • Use methods like GREMLIN or EVcouplings to predict residue contacts

• Metabolic modeling:

  • Incorporate MMP0079 into a genome-scale metabolic model of M. maripaludis

  • Predict metabolic flux through the pyrimidine pathway

  • Simulate the effects of MMP0079 deletion or overexpression

These computational approaches provide testable hypotheses and guide experimental design, offering insights into MMP0079 that might be difficult to obtain through experimental methods alone.

What is the impact of environmental conditions on MMP0079 expression and activity?

Understanding how environmental factors affect MMP0079 expression and activity requires systematic investigation:

• Expression analysis under varying conditions:

  • Growth temperature variation (given M. maripaludis is mesophilic, test 20-45°C range)

  • Different carbon and nitrogen sources

  • Hydrogen limitation (as M. maripaludis is hydrogenotrophic)

  • Nutrient limitation and starvation

  • Stress conditions (pH, osmotic, oxidative stress)

• Activity assays under different physicochemical conditions:

  • Determine temperature optimum and stability

  • Establish pH profile for enzyme activity

  • Test effects of various salt concentrations and ionic species

  • Examine metal ion requirements and inhibition

• Adaptation experiments:

  • Investigate how long-term growth under specific conditions affects MMP0079 expression

  • Compare activity and expression between wild isolates from different environments

  • Examine regulatory adaptations in experimentally evolved strains

• Comparative analysis with MMP1492:

  • Determine if the two paralogs show differential expression patterns under varying conditions

  • Investigate whether they have different temperature, pH, or substrate optima

  • Test the hypothesis that gene duplication allows specialization for different environmental niches

M. maripaludis, as a mesophilic archaeon, has adapted to moderate temperature environments, unlike its thermophilic relatives like Methanocaldococcus jannaschii . This adaptation likely extends to enzymes like MMP0079, with expected optimal activity around 35-40°C. Understanding how environmental conditions affect MMP0079 will provide insights into archaeal adaptations to specific ecological niches.

How can MMP0079 be used in synthetic biology applications?

Potential applications of recombinant MMP0079 in synthetic biology include:

• In vitro nucleotide synthesis:

  • Development of enzymatic cascades for the synthesis of modified nucleotides

  • Production of isotopically labeled nucleotides for NMR studies

  • Creation of nucleotide analogs for pharmaceutical applications

• Biosensor development:

  • Engineering MMP0079-based biosensors for detecting orotate or PRPP in biological samples

  • Creating coupled enzyme assays for high-throughput screening applications

  • Developing whole-cell biosensors for monitoring pyrimidine metabolism

• Metabolic engineering:

  • Introduction of archaeal pyrimidine pathways into bacterial or eukaryotic hosts

  • Enhancement of nucleotide production in industrial strains

  • Engineering of nucleotide salvage pathways for efficient utilization of precursors

• Enzyme evolution and engineering:

  • Directed evolution of MMP0079 for improved catalytic efficiency

  • Engineering of substrate specificity to accept non-natural substrates

  • Adaptation of the enzyme for functioning in non-native hosts or conditions

• Comparative biochemistry educational tools:

  • Use of MMP0079 as a model system for teaching archaeal enzymology

  • Development of comparative biochemistry exercises examining enzymes from all three domains of life

For such applications, researchers should consider the unique properties of archaeal enzymes, including potential differences in folding, cofactor requirements, and optimal reaction conditions. The mesophilic nature of M. maripaludis makes MMP0079 particularly suitable for applications at moderate temperatures, unlike enzymes from hyperthermophilic archaea that may require extreme conditions .

What are emerging technologies for studying archaeal enzymes like MMP0079?

Several cutting-edge technologies are poised to advance our understanding of archaeal enzymes like MMP0079:

• Single-molecule enzymology:

  • Apply techniques like TIRFM (Total Internal Reflection Fluorescence Microscopy) to observe individual enzyme molecules

  • Use FRET (Förster Resonance Energy Transfer) to monitor conformational changes during catalysis

  • Implement magnetic tweezers or optical traps to study enzyme mechanics at the single-molecule level

• Cryo-electron microscopy:

  • Determine high-resolution structures of MMP0079 alone and in complex with substrates or other proteins

  • Visualize MMP0079 in the context of larger macromolecular assemblies

  • Map conformational states throughout the catalytic cycle

• Time-resolved structural methods:

  • Apply time-resolved X-ray crystallography to capture intermediate states

  • Use time-resolved spectroscopy to follow reaction progression

  • Implement temperature-jump or rapid mixing techniques to synchronize reactions

• Systems biology approaches:

  • Integrate multi-omics data (genomics, transcriptomics, proteomics, metabolomics) to understand MMP0079 in its cellular context

  • Develop kinetic models of the pyrimidine biosynthesis pathway

  • Apply flux analysis to quantify metabolic flow through pathways involving MMP0079

• CRISPR-based technologies for archaeal systems:

  • Develop improved gene editing tools for Methanococcus maripaludis

  • Create CRISPRi systems for tunable gene repression

  • Implement CRISPR-based imaging to track protein localization in vivo

These technologies will help address fundamental questions about MMP0079 function and potentially reveal unexpected roles or regulatory mechanisms not captured by traditional biochemical approaches.

What unresolved questions remain about MMP0079 and archaeal PyrE proteins?

Despite advances in our understanding of archaeal metabolism, several key questions about MMP0079 and related PyrE proteins remain unanswered:

• Evolutionary history and specialization:

  • Why do some archaea, including M. maripaludis, maintain two PyrE paralogs while others have only one?

  • What selective pressures drive the conservation of these paralogs?

  • How have these enzymes evolved across the three domains of life?

• Regulatory networks:

  • How is MMP0079 expression coordinated with other enzymes in nucleotide metabolism?

  • What transcription factors control its expression?

  • Are there post-translational modifications that regulate its activity?

• Structural adaptations:

  • What structural features distinguish archaeal PyrE proteins from their bacterial and eukaryotic counterparts?

  • How have mesophilic archaeal enzymes like MMP0079 adapted compared to thermophilic homologs?

  • What determines substrate specificity and catalytic efficiency?

• Cellular role beyond canonical function:

  • Does MMP0079 participate in protein-protein interactions or moonlighting functions?

  • Are there alternative substrates or reaction pathways?

  • How does the enzyme contribute to metabolic homeostasis?

• Technological applications:

  • Can archaeal PyrE proteins be engineered for biotechnological applications?

  • Do these enzymes have advantageous properties for synthetic biology?

  • Could inhibitors of these enzymes have antimicrobial potential against pathogenic archaea?

Addressing these questions will require interdisciplinary approaches combining structural biology, biochemistry, genetics, and systems biology. The genetic tractability of M. maripaludis makes it an excellent model system for such investigations .