Recombinant Opitutus terrae Peptide deformylase (def)

What is Opitutus terrae and how is it taxonomically classified?

Opitutus terrae is an obligately anaerobic bacterium isolated from rice paddy soil microcosms. It belongs to the division 'Verrucomicrobia', though recent phylogenetic studies suggest it may be more accurately placed in the proposed phylum Kiritimatiellaeota. The cells are cocci-shaped and motile via a flagellum. Neither catalase nor oxidase activities are present in these organisms .

Taxonomically, Opitutus terrae forms a distinct lineage that has evolved separately from other Verrucomicrobia members, as demonstrated by phylogenetic analyses of 16S rRNA genes and protein-coding sequences. This evolutionary divergence is reflected in the percentage of positive BLAST hits between Opitutus terrae and established Verrucomicrobia members being below 10%, whereas strains representing the five main lineages of Verrucomicrobia display values of 40% or above .

What is peptide deformylase (def) and what is its biological function?

Peptide deformylase is an essential Fe²⁺ metalloenzyme that catalyzes the removal of N-terminal formyl groups from nascent polypeptides in eubacteria. In prokaryotic protein synthesis, translation initiates with a formylated methionine, and the removal of this formyl group is a critical post-translational modification required for proper protein maturation and function .

The enzyme plays a fundamental role in bacterial protein synthesis, making it an attractive target for antibacterial drug discovery. The essential nature of this enzyme has been demonstrated in various bacterial species through gene knockout studies, where loss of deformylase activity typically results in non-viable organisms unless compensatory mechanisms are present .

How does the substrate specificity of peptide deformylase function?

Despite being considered a broad-specificity enzyme, peptide deformylase exhibits varying degrees of efficiency in deformylating different peptide sequences. The E. coli enzyme, for example, has a consensus sequence preference of formyl-Met-X-Z-Tyr, where X represents any amino acid except for aspartate and glutamate, and Z can be lysine, arginine, tyrosine, or phenylalanine .

Additionally, the enzyme demonstrates selectivity for N-formyl over N-acetyl groups, with both electronic and steric factors contributing to this specificity. The enzyme can also efficiently deformylate formyl-Phe-Tyr-(Phe/Tyr) peptides, indicating flexibility in recognizing certain non-methionine N-terminal residues .

The substrate specificity can be systematically examined using combinatorial methods involving peptide libraries that contain all possible N-terminally formylated peptides of a given length. By treating these libraries with the deformylase and identifying which peptides are most efficiently processed, researchers can establish the sequence preferences of the enzyme .

What are the optimal expression systems for producing recombinant Opitutus terrae peptide deformylase?

For recombinant expression of Opitutus terrae peptide deformylase, E. coli-based expression systems are typically employed. The expression strategy should address several key considerations specific to this anaerobic organism's proteins:

• Vector selection: pET-series vectors with T7 promoter systems are commonly used for high-level expression of recombinant proteins.

• Expression conditions: Since Opitutus terrae is an obligate anaerobe, its proteins may be sensitive to oxidative conditions. Expression should therefore be conducted at lower temperatures (16-20°C) with reduced inducer concentrations to allow proper folding and minimize inclusion body formation .

• Host strain selection: E. coli strains like BL21(DE3) or Rosetta(DE3) for rare codon optimization may be necessary, as the high G+C content (74 mol%) of Opitutus terrae's genomic DNA suggests potential codon usage differences .

• Co-expression strategies: Since peptide deformylase is an Fe²⁺ metalloenzyme, co-expression with iron-sulfur cluster assembly proteins or supplementation with iron in the growth medium may improve the yield of active enzyme.

What purification strategies yield the highest activity for recombinant peptide deformylase?

A multi-step purification strategy optimized for maintaining enzymatic activity would include:

Throughout the purification process, it is critical to:

• Maintain reducing conditions to prevent oxidation of the Fe²⁺ cofactor

• Work under anaerobic or low-oxygen conditions whenever possible

• Include glycerol (10-20%) in buffers to enhance stability

• Avoid EDTA or other chelating agents that may sequester the metal cofactor

How can enzyme activity be preserved during storage and handling?

To maximize stability and retain activity during storage:

• Buffer composition: Store in 50 mM HEPES or phosphate buffer, pH 7.0-7.5, containing 100-200 mM NaCl, 10-20% glycerol, and 1-5 mM DTT or β-mercaptoethanol.

• Metal supplementation: Include 0.1-0.2 mM ferrous ammonium sulfate in storage buffers, as the Fe²⁺ cofactor is essential for activity.

• Storage temperature: Store at -80°C for long-term preservation or at -20°C for short-term storage in small aliquots to avoid repeated freeze-thaw cycles.

• Handling considerations: Work under anaerobic conditions or use an oxygen-scavenging system, particularly important for proteins from obligate anaerobes like Opitutus terrae .

What are the established methods for measuring peptide deformylase activity?

Several approaches can be employed to measure peptide deformylase activity:

• Formate dehydrogenase-coupled assays: This spectrophotometric method couples the release of formate during deformylation to the reduction of NAD⁺ by formate dehydrogenase, allowing continuous monitoring of activity via absorbance changes at 340 nm.

• HPLC-based assays: This approach involves separation and quantification of formylated substrate and deformylated product peptides using reverse-phase HPLC.

• Fluorescence-based assays: Using fluorogenic peptide substrates where deformylation results in a change in fluorescence properties.

• Colorimetric methods: Detection of released formate using colorimetric reagents that form colored complexes with formate.

For optimal results with Opitutus terrae peptide deformylase, the assays should be conducted under anaerobic conditions, given the obligate anaerobic nature of the source organism .

How can substrate specificity be comprehensively analyzed?

A systematic analysis of substrate specificity can be performed using the following methodology:

• Peptide library construction: Develop a combinatorial peptide library containing diverse N-terminally formylated peptides on solid support (e.g., TentaGel resin), with a unique peptide sequence on each resin bead .

• Limited enzymatic treatment: Expose the peptide library to the recombinant Opitutus terrae peptide deformylase under conditions that result in partial deformylation, allowing the most preferred substrates to be processed first .

• Identification of deformylated peptides: Use an enzyme-linked assay to identify and isolate beads containing deformylated peptides .

• Sequence analysis: Perform mass spectrometry (e.g., MALDI-TOF MS) to determine the sequence of the preferentially deformylated peptides .

• Consensus sequence determination: Analyze the sequence data to identify patterns or motifs that define the substrate preference of the enzyme.

This approach has been successfully used with E. coli peptide deformylase to identify its consensus sequence preference (formyl-Met-X-Z-Tyr) .

What kinetic parameters should be determined and how?

A comprehensive kinetic characterization should include:

For accurate determination of these parameters:

• Use initial rate conditions (< 10% substrate conversion)

• Ensure enzyme concentration is significantly below substrate concentration

• Maintain anaerobic conditions throughout the assay

• Include appropriate controls for background reactions and instrument drift

How does the presence of multiple deformylase genes affect bacterial physiology?

Some bacteria possess multiple peptide deformylase genes with distinct functional roles. In Bacillus subtilis, two deformylase genes (def and ykrB) have been identified, with YkrB appearing to be the predominant deformylase in normal growth conditions .

This gene duplication has several implications:

• Functional redundancy: Either def or ykrB can support growth in rich medium, with each being individually dispensable but at least one required for viability .

• Differential expression: The two genes may be expressed under different conditions or growth phases, allowing adaptability to environmental changes.

• Target for antimicrobials: Both gene products have been shown to be cellular targets for deformylase inhibitors such as actinonin, with similar inhibition efficacy in biochemical assays .

• Resistance development: Interestingly, the presence of dual deformylase genes does not increase actinonin-resistance frequency compared to B. subtilis mutants carrying only one deformylase gene .

Whether Opitutus terrae possesses multiple deformylase genes remains to be determined through genomic analysis, but the functional implications would likely be similar if gene duplication exists.

How do site-directed mutations affect catalytic activity and substrate specificity?

Site-directed mutagenesis studies of peptide deformylases have revealed several key structure-function relationships:

• Metal-binding residues: Mutations in the conserved HEXXH motif, which coordinates the Fe²⁺ cofactor, typically result in complete loss of catalytic activity, highlighting the essential role of the metal center.

• Substrate binding pocket: Alterations in residues lining the substrate binding site can significantly affect:

  • Substrate specificity profiles

  • Catalytic efficiency (kcat/Km)

  • Binding affinity for different N-terminal sequences

• Catalytic mechanism residues: Mutations of residues involved in the catalytic mechanism, such as those participating in hydrogen bonding networks or stabilizing transition states, can reveal rate-limiting steps in the reaction.

A methodical approach to mutagenesis studies would involve:

• Alanine scanning of the active site residues

• Conservative mutations to probe specific chemical interactions

• Creation of chimeric enzymes combining domains from different bacterial deformylases

• Correlation of mutational effects with structural changes determined by X-ray crystallography

How do environmental factors affect enzyme activity and stability?

Given that Opitutus terrae is an obligate anaerobe isolated from rice paddy soil , its peptide deformylase would be expected to show adaptations to this specific environment:

The enzyme's adaptation to anaerobic conditions would likely manifest as increased sensitivity to oxidative inactivation compared to deformylases from aerobic organisms, necessitating careful handling under reducing conditions .

How do various inhibitors affect Opitutus terrae peptide deformylase activity?

Peptide deformylase inhibitors represent a promising class of antibacterial agents. A systematic approach to characterizing inhibition would include:

• Natural product inhibitors: Actinonin, the prototype natural product inhibitor, has been shown to effectively inhibit various bacterial peptide deformylases, including those from B. subtilis . Testing against Opitutus terrae peptide deformylase would establish baseline inhibition parameters.

• Synthetic inhibitors: Various classes of synthetic inhibitors have been developed, including:

  • Hydroxamate-based inhibitors

  • Reverse hydroxamate inhibitors

  • N-formyl-N-hydroxylamine derivatives

  • Metalloprotease inhibitors adapted for peptide deformylase

• Structure-activity relationships: By testing series of related compounds, structure-activity relationships can be established to guide the design of selective inhibitors.

The inhibition profile would provide valuable information about:

• The structural features of the active site

• Unique characteristics of the Opitutus terrae enzyme compared to other bacterial deformylases

• Potential selectivity that could be exploited for targeted antimicrobial development

What are the most effective methods for screening novel inhibitors?

A comprehensive inhibitor screening strategy would employ multiple complementary approaches:

• High-throughput biochemical assays:

  • Fluorescence-based assays in 384 or 1536-well format

  • Formate detection assays adapted for high-throughput screening

  • Thermal shift assays to identify compounds that stabilize the enzyme structure

• Fragment-based screening:

  • NMR-based fragment screening to identify low molecular weight binders

  • X-ray crystallography to confirm binding modes

  • Fragment linking and growing to develop high-affinity inhibitors

• In silico approaches:

  • Structure-based virtual screening using the crystal structure or homology model

  • Pharmacophore modeling based on known inhibitors

  • Molecular dynamics simulations to identify transient binding pockets

• Phenotypic validation:

  • Growth inhibition assays with bacteria expressing Opitutus terrae peptide deformylase

  • Target engagement studies to confirm that growth inhibition correlates with enzyme inhibition

These approaches should be conducted under anaerobic conditions when possible to maintain the native state of the enzyme .

How can inhibitor selectivity between bacterial deformylases be achieved?

Developing selective inhibitors for specific bacterial peptide deformylases requires:

• Comparative structural analysis:

  • Crystal structures or homology models of multiple bacterial deformylases

  • Identification of unique structural features in the Opitutus terrae enzyme

  • Analysis of active site architecture differences

• Differential inhibition profiling:

  • Side-by-side testing of inhibitor panels against multiple bacterial deformylases

  • Calculation of selectivity indices (ratio of IC₅₀ values)

  • Identification of inhibitor scaffolds with inherent selectivity

• Structure-guided optimization:

  • Targeting non-conserved residues near the active site

  • Exploiting differences in substrate binding pockets

  • Introducing structural elements that create steric clashes specifically in non-target enzymes

• Exploiting unique features of Opitutus terrae:

  • Its adaptation to anaerobic environments may have led to unique structural features

  • Potential differences in metal coordination that could be targeted

  • Distinct substrate preferences that could inform inhibitor design

What spectroscopic methods are most informative for studying metal coordination?

The metal center is crucial for peptide deformylase activity, making its characterization essential. Recommended spectroscopic approaches include:

• X-ray absorption spectroscopy (XAS):

  • Extended X-ray absorption fine structure (EXAFS) to determine Fe-ligand distances

  • X-ray absorption near edge structure (XANES) to determine Fe oxidation state

  • Data collection under anaerobic conditions to prevent oxidation

• Electron paramagnetic resonance (EPR):

  • Characterization of the Fe³⁺ form (which is paramagnetic)

  • Spin-trapping experiments to detect reactive intermediates

  • Temperature-dependent measurements to study magnetic properties

• Mössbauer spectroscopy:

  • Direct probe of the iron electronic environment

  • Differentiation between Fe²⁺ and Fe³⁺ states

  • Analysis of ligand effects on the iron center

• Resonance Raman spectroscopy:

  • Investigation of metal-ligand vibrations

  • Study of structural changes upon substrate binding

  • Characterization of inhibitor interactions with the metal center

These techniques, particularly when used in combination, can provide detailed information about the coordination environment and oxidation state of the iron cofactor, which is critical for understanding the catalytic mechanism .

How can crystallography and computational modeling be integrated for mechanistic studies?

A comprehensive approach combining experimental crystallography with computational modeling offers powerful insights:

• X-ray crystallography:

  • Determination of enzyme structure at high resolution

  • Co-crystallization with substrates or substrate analogs

  • Collection of structures representing different states of the catalytic cycle

• Computational methods:

  • Molecular dynamics simulations to explore conformational flexibility

  • Quantum mechanics/molecular mechanics (QM/MM) calculations to model reaction mechanisms

  • Docking studies to predict binding modes of substrates and inhibitors

• Integration approaches:

  • Using crystallographic structures as starting points for computational studies

  • Validating computational predictions with new crystal structures

  • Designing mutations based on computational hypotheses and testing experimentally

• Specific applications:

  • Elucidating the proton transfer pathway during catalysis

  • Investigating the role of water molecules in the active site

  • Understanding substrate specificity at the atomic level

This integrated approach is particularly valuable for peptide deformylase, where the catalytic mechanism involves subtle electronic and steric effects that are difficult to capture with any single technique .

How does Opitutus terrae peptide deformylase compare to other bacterial deformylases?

A comparative analysis would reveal evolutionary adaptations and potential functional specializations:

The anaerobic lifestyle of Opitutus terrae may have driven unique adaptations in its peptide deformylase, particularly in terms of oxygen sensitivity and substrate preferences that reflect the organism's metabolic capabilities .

What evolutionary insights can be gained from phylogenetic analysis?

Phylogenetic analysis of peptide deformylases can reveal:

• Evolutionary trajectory:

  • Placement of Opitutus terrae peptide deformylase within the broader PDF family tree

  • Identification of closely related enzymes from other anaerobic bacteria

  • Analysis of selective pressures acting on different regions of the protein

• Gene duplication events:

  • Evidence for potential gene duplication in Opitutus terrae, similar to that observed in B. subtilis

  • Timing of duplication events relative to speciation events

  • Functional divergence patterns following duplication

• Horizontal gene transfer:

  • Potential instances of horizontal gene transfer involving deformylase genes

  • Analysis of anomalous phylogenetic placements or GC content

  • Implications for the spread of antimicrobial resistance mechanisms

• Correlation with ecological adaptations:

  • Relationship between deformylase evolution and adaptation to specific ecological niches

  • Patterns of sequence conservation in organisms with similar lifestyles

  • Connection to the evolution of protein synthesis machinery in different bacterial lineages

This phylogenetic context would provide a framework for understanding the unique features of Opitutus terrae peptide deformylase in relation to its evolutionary history and ecological role.