Recombinant Prochlorococcus marinus subsp. pastoris UDP-N-acetylmuramoylalanine--D-glutamate ligase (murD)

What is the biological significance of MurD in Prochlorococcus marinus?

MurD (UDP-N-acetylmuramoylalanine--D-glutamate ligase) is a cytoplasmic enzyme critical for peptidoglycan biosynthesis in bacteria. In Prochlorococcus marinus, this enzyme catalyzes the addition of D-glutamate to UDP-N-acetylmuramoyl-L-alanine (UMA), representing the second amino acid addition in the peptidoglycan precursor synthesis pathway . The significance of studying this enzyme in Prochlorococcus marinus lies in understanding cell wall formation in one of the most abundant photosynthetic organisms in marine ecosystems. As a cyanobacterium responsible for a significant portion of oceanic primary production, understanding its cell wall biosynthesis provides insights into both fundamental bacterial physiology and potential ecological adaptations.

How does the structure of MurD from Prochlorococcus marinus compare to other bacterial homologs?

The MurD enzyme from Prochlorococcus marinus follows the general structural organization observed in other bacterial MurD enzymes, comprising three domains with nucleotide-binding folds: N-terminal, central, and C-terminal domains . The N-terminal and C-terminal domains display a dinucleotide-binding Rossmann fold, while the central domain exhibits a mononucleotide-binding fold similar to that found in the GTPase family .

What are the optimal conditions for culturing Prochlorococcus marinus for recombinant protein studies?

Culturing Prochlorococcus marinus for recombinant protein studies requires careful consideration of growth media and conditions. Based on established protocols, researchers should consider the following approach:

For optimal results, cultures should be maintained in exponential growth phase through regular transfer to fresh media. When scaling up for protein production, consider that different Prochlorococcus strains show variable growth rates in these media, with high-light adapted strains typically growing faster . The relative growth rates of different strains in various media should be empirically determined for each specific strain being used for recombinant protein expression.

What expression systems are most effective for recombinant Prochlorococcus MurD production?

While the search results don't directly address expression systems for Prochlorococcus MurD, effective heterologous expression typically employs E. coli-based systems with specific modifications to accommodate cyanobacterial proteins. The recommended methodological approach includes:

• Vector selection: pET-based expression vectors containing T7 promoter systems offer strong, inducible expression for enzymatic studies.

• Host strain optimization: E. coli BL21(DE3) derivatives with modifications such as rare codon supplementation (e.g., Rosetta strains) may improve expression of cyanobacterial genes that contain rare codons.

• Expression conditions: Induction at lower temperatures (16-20°C) with reduced IPTG concentrations (0.1-0.5 mM) typically enhances soluble protein production for enzymatic studies.

• Codon optimization: Synthesizing the Prochlorococcus murD gene with codons optimized for E. coli expression can significantly improve protein yields.

• Fusion tags: N-terminal His6 or MBP fusion tags facilitate purification while potentially enhancing solubility.

The selection of expression parameters should be experimentally determined through small-scale expression trials analyzing soluble versus insoluble protein fractions before scaling up production.

What purification strategy provides the highest yield and purity for recombinant MurD?

A multistep purification strategy is recommended for isolating high-purity recombinant MurD:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin for His-tagged MurD, with binding in 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole, and elution with an imidazole gradient (50-250 mM).

• Intermediate purification: Ion exchange chromatography using either anion (Q-Sepharose) or cation (SP-Sepharose) exchangers depending on the calculated pI of the MurD construct.

• Polishing step: Size exclusion chromatography using Superdex 200 in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM DTT to separate monomeric enzyme from aggregates and remove remaining contaminants.

• Quality assessment: SDS-PAGE analysis (>95% purity), Western blotting, and activity assays measuring ATP consumption and D-glutamate incorporation into UMA.

For enzymatic studies, the final preparation should be concentrated to 1-5 mg/mL, flash-frozen in liquid nitrogen with 10% glycerol, and stored at -80°C in single-use aliquots.

How can researchers effectively perform crystallographic studies of Prochlorococcus MurD?

Crystallization of Prochlorococcus MurD requires careful consideration of protein conformational states and ligand binding. Based on structural studies of bacterial MurD enzymes, researchers should employ the following methodological approach:

• Protein preparation: Highly purified MurD (>98% purity) at 5-10 mg/mL in a minimal buffer (e.g., 10 mM Tris-HCl pH 7.5, 50 mM NaCl).

• Co-crystallization approach: Include substrate UMA at 2-5 mM concentration to stabilize the enzyme structure, as demonstrated in previous MurD structural studies .

• Initial screening: Employ commercial sparse matrix screens at multiple temperatures (4°C, 16°C, and 20°C) with both hanging drop and sitting drop vapor diffusion methods.

• Optimization strategies: Based on initial hits, optimize promising conditions by varying:

  • Protein concentration (3-15 mg/mL)

  • Precipitant concentration (±5-10%)

  • pH (±0.5 units)

  • Additives (particularly divalent cations like Mg²⁺)

• Data collection considerations: Crystals should be cryoprotected using mother liquor supplemented with 20-25% glycerol or ethylene glycol before flash-cooling in liquid nitrogen for synchrotron data collection.

The presence of substrate UMA has been shown to facilitate crystallization by stabilizing domain orientation, as demonstrated in the 1.9 Å resolution structure of MurD with bound UMA .

What assays provide the most reliable quantification of MurD enzymatic activity?

MurD activity can be reliably quantified using several complementary approaches:

• Coupled spectrophotometric assay: ATP hydrolysis during the reaction can be coupled to NADH oxidation through pyruvate kinase and lactate dehydrogenase, allowing continuous monitoring at 340 nm.

• Radioactive assay: Using ¹⁴C-labeled D-glutamate or ³²P-labeled ATP to trace product formation, followed by separation via thin-layer chromatography or paper electrophoresis.

• HPLC-based assay: Direct measurement of UDP-MurNAc-dipeptide formation using reverse-phase HPLC with UV detection at 254 nm for the nucleotide moiety.

• Malachite green phosphate assay: Measuring released inorganic phosphate from ATP hydrolysis as an indirect reporter of enzymatic activity.

For kinetic characterization, steady-state kinetic studies should be performed varying each substrate (UMA, D-glutamate, and ATP) while maintaining others at saturating concentrations to determine Km and kcat values . This approach provides valuable information about the reaction mechanism and can identify rate-limiting steps in the catalytic cycle.

How does ATP binding affect the conformational dynamics of MurD?

ATP binding induces significant conformational changes in MurD that are critical for catalytic activity. Multiple protein structures and molecular dynamics simulations reveal:

• Domain movement: ATP binding promotes closure of the C-terminal domain toward the central domain, bringing catalytic residues into proper orientation for facilitating D-glutamate incorporation .

• Energy landscape: Molecular dynamics and Off-Path Simulation (OPS) techniques have demonstrated that the closed conformation of MurD represents the lowest energy state when ATP is bound .

• Conformational equilibrium: There exists an equilibrium of protein structures between the open (unbound) and closed (ATP-bound) states, with intermediate conformations readily accessible .

• Structural implications: ATP binding residues, primarily located in the central domain, have been identified through comparison with known NTP complexes and simulation studies .

The N-terminal domain and central domain remain relatively fixed during catalysis, while the C-terminal domain undergoes the largest conformational changes, as demonstrated by RMSD analysis during molecular dynamics simulations (with RMSD values for C-terminal movement exceeding those of N-terminal domains) .

What strategies are most effective for designing inhibitors targeting Prochlorococcus MurD?

The most effective inhibitor design strategies for MurD utilize understanding of the enzyme's conformational flexibility and binding site characteristics:

• Multiple conformation targeting: Selection of multiple protein conformations (closed, open, and intermediate states) for virtual screening enhances the discovery of diverse inhibitor classes .

• Structure-based pharmacophore development: Development of pharmacophore models based on key interaction features of the ATP-binding site, with particular attention to conserved binding residues .

• Fragment-based approaches: Identification of fragment hits that bind to different sub-pockets within the ATP-binding site, followed by fragment growing or linking strategies.

• Molecular dynamics validation: Validation of potential inhibitors through molecular dynamics simulations to assess binding stability across different enzyme conformations .

This multi-conformational approach has successfully identified aminothiazole-based compounds (compound 3) with MurD inhibitory activity in the micromolar range, demonstrating efficacy of this strategy .

How can researchers conduct effective inhibitor screening assays for MurD?

Effective inhibitor screening for MurD should employ a multi-tiered approach:

• Primary screening:

  • Spectrophotometric coupled assay in 96-well format (Z' > 0.7)

  • Compound concentration: 10-50 μM

  • Controls: Known MurD inhibitors and DMSO vehicle

  • Cutoff: >50% inhibition for hit selection

• Secondary validation:

  • Dose-response determination (IC₅₀)

  • Counterscreening against coupling enzymes to eliminate false positives

  • Kinetic characterization to determine inhibition modality (competitive, noncompetitive, or uncompetitive)

• Mechanistic characterization:

  • Steady-state kinetic studies varying substrate concentrations to distinguish ATP-competitive from D-glutamate-competitive inhibitors

  • Thermal shift assays to evaluate compound binding and protein stabilization

• Biophysical validation:

  • Surface plasmon resonance or isothermal titration calorimetry to determine binding constants and thermodynamic parameters

  • X-ray crystallography to confirm binding mode and interaction patterns

This approach has successfully identified dual MurC/MurD inhibitors, demonstrating cross-inhibition potential within the Mur ligase family .

How do inhibitor binding modes differ between open and closed conformations of MurD?

Inhibitor binding modes show significant differences between open and closed MurD conformations:

• Closed conformation (2UAG structure):

  • More stable inhibitor binding with lower positional fluctuation

  • Primarily central domain interactions with partial C-terminal contacts

  • Restricted binding pocket with well-defined interaction patterns

  • Higher energetic favorability for inhibitor retention

• Intermediate conformations (1EEH-2UAG and 1E0D-2UAG):

  • Dynamic inhibitor binding with greater positional variability

  • Potential for unique interaction patterns not accessible in fully closed states

  • Domain movement during binding can induce inhibitor repositioning

• Open conformation:

  • Larger binding pocket with increased solvent accessibility

  • Potential for capturing different inhibitor chemotypes

  • Greater C-terminal domain mobility affecting binding stability

Molecular dynamics simulations revealed that with aminothiazole compound 3, the C-terminal domain of the intermediate 1EEH-2UAG MurD structure exhibited significant opening movement during simulation, while more stable inhibitor positioning was observed in the closed 2UAG structure .

How does recombinant expression affect post-translational modifications of Prochlorococcus MurD?

When expressing recombinant Prochlorococcus MurD, researchers should consider:

• Lysine carbamylation: Crystallographic studies of MurD have identified carbamylated lysine residue 198 (KCX198), which may influence catalytic activity . In recombinant expression systems, this modification may occur spontaneously but at potentially different rates than in the native environment.

• Phosphorylation: While not explicitly mentioned in the search results for Prochlorococcus MurD, bacterial protein phosphorylation can affect enzyme activity and regulation. Heterologous expression systems may lack the specific kinases present in Prochlorococcus.

• Methodology for assessment: Researchers should employ mass spectrometry-based approaches to comprehensively characterize post-translational modifications in recombinant MurD:

  • Bottom-up proteomics with enrichment strategies for modified peptides

  • Intact protein mass analysis to determine modification stoichiometry

  • Site-directed mutagenesis to assess the functional impact of specific modifications

Recent structural studies have shown that KCX198 can exist in different configurations, implying functional flexibility that may be relevant to enzyme regulation .

What are the key differences in kinetic parameters between MurD from Prochlorococcus and other bacterial species?

Comparative kinetic analysis between Prochlorococcus MurD and homologs from other bacteria reveals important functional distinctions:

These kinetic differences reflect evolutionary adaptations to specific environmental niches and growth requirements. When conducting comparative kinetic studies, researchers should standardize reaction conditions and employ the same methodology across all enzyme variants to ensure valid comparisons.

How can molecular dynamics simulations enhance understanding of Prochlorococcus MurD function?

Molecular dynamics (MD) simulations provide valuable insights into MurD function beyond static crystal structures:

• Domain motion analysis: MD simulations reveal the dynamic movement of the C-terminal domain during the catalytic cycle, with RMSD analysis quantifying the extent and direction of this movement .

• Energy landscape mapping: Techniques such as Targeted Molecular Dynamics (TMD) and Off-Path Simulation (OPS) identify energetically favorable conformational states and transition pathways between open and closed forms .

• Ligand binding dynamics: Simulation of inhibitor-enzyme complexes reveals differential binding stability across different conformational states, informing inhibitor design strategies .

• Methodological approach:

  • System preparation using CHARMM-GUI for protein manipulation and solvation

  • Parameter assignment using CHARMM force field for protein and CGenFF for ligands

  • Production simulations of at least 10 ns following equilibration procedures

  • Analysis of domain movements by RMSD calculation after central domain alignment

MD simulations have demonstrated that the closed conformation (2UAG structure) corresponds to the lowest energy state on the investigated OPS energy landscape, while intermediate structures from different pathways (1E0D-2UAG and 1EEH-2UAG) show distinct behaviors when complexed with inhibitors .

What are common expression challenges specific to Prochlorococcus proteins and how can they be overcome?

Expression of recombinant proteins from Prochlorococcus presents several challenges:

• Codon usage bias: Prochlorococcus genomes have distinctive codon preferences that differ from common expression hosts like E. coli.

  • Solution: Use codon-optimized synthetic genes or expression hosts supplemented with rare tRNAs (e.g., Rosetta strains).

• Protein folding in heterologous hosts: Cyanobacterial proteins may require specific chaperones absent in E. coli.

  • Solution: Co-express molecular chaperones (GroEL/ES, DnaK systems) or use speciality strains like Arctic Express with cold-adapted chaperones.

• Toxicity to host cells: Some cyanobacterial proteins may be toxic when overexpressed.

  • Solution: Use tightly regulated expression systems (e.g., pET with T7-lysozyme co-expression) or lower-copy-number vectors with moderate promoters.

• Membrane association: Even cytoplasmic proteins may have membrane interactions in cyanobacteria.

  • Solution: Optimize lysis conditions with various detergents (0.1-1% Triton X-100, CHAPS, or n-Dodecyl β-D-maltoside) to improve solubility.

• Methodological recommendations:

  • Test multiple construct designs with varying tag positions and linker sequences

  • Optimize induction conditions (temperature, inducer concentration, duration)

  • Screen multiple E. coli strains in parallel small-scale expression trials

How can researchers effectively handle the instability of UDP-MurNAc-peptide intermediates in MurD assays?

Working with UDP-MurNAc-peptide intermediates presents stability challenges that can be addressed through:

• Storage considerations:

  • Maintain stock solutions of UMA substrate at -80°C in small single-use aliquots

  • Prepare fresh working solutions daily from frozen stocks

  • Add stabilizers like 10% glycerol and 1 mM DTT to prevent degradation

• Handling during assays:

  • Keep all reaction components on ice until immediately before the assay

  • Use temperature-controlled reaction blocks/plates for consistent results

  • Include controls to monitor substrate stability over the assay timeframe

• Analysis methods:

  • For chromatographic analysis, develop rapid sample processing workflows to minimize degradation

  • Consider using ion-pairing reagents in HPLC methods to improve peak shape and resolution

  • Develop LC-MS methods for unambiguous identification of reaction products and degradation products

• Special considerations for kinetic studies:

  • Account for potential substrate degradation in kinetic calculations

  • Design time-course experiments to ensure linearity during the measurement period

  • Include additional controls at different substrate concentrations to identify concentration-dependent degradation effects

These methodological approaches minimize experimental variability and improve the reproducibility of MurD activity measurements.

How might comparative analysis of MurD across Prochlorococcus ecotypes inform evolutionary adaptation to marine environments?

Investigating MurD across Prochlorococcus ecotypes presents opportunities to:

• Correlate enzyme kinetics with environmental adaptation: The various Prochlorococcus strains (high-light vs. low-light adapted; MED4ax, MIT9312ax, NATL2a, SS120, MIT9211, MIT9313ax) show different growth characteristics in various media . These adaptations may extend to differences in cell wall synthesis enzymes like MurD.

• Methodological approach for comparative studies:

  • Clone and express murD genes from multiple ecotypes (high-light and low-light adapted)

  • Conduct parallel biochemical characterization under identical conditions

  • Analyze temperature dependence profiles reflecting ocean depth adaptation

  • Compare substrate affinities in relation to cellular metabolite pools

• Structure-function relationships: Sequence variations in MurD across ecotypes likely reflect adaptations to different ocean environments, potentially affecting:

  • Substrate binding pocket architecture

  • Domain flexibility and conformational dynamics

  • Catalytic efficiency at different temperatures

  • Susceptibility to inhibition

• Evolutionary insights: Phylogenetic analysis of MurD across Prochlorococcus ecotypes could reveal patterns of adaptive evolution in peptidoglycan biosynthesis pathways, possibly correlating with genome streamlining observed in these organisms.

Such comparative approaches would provide novel insights into how essential cellular processes adapt to specific marine environmental niches.

What are the implications of MurD inhibition for marine microbial ecology and biogeochemical cycling?

The implications of MurD inhibition extend beyond basic enzymology to broader ecological considerations:

• Ecological specificity: Inhibitors with differential activity against MurD from various marine bacteria could potentially affect community composition by selectively inhibiting certain species or strains.

• Methodological considerations for ecological studies:

  • Develop reporter strains with fluorescent markers linked to cell wall stress responses

  • Conduct microcosm experiments with natural communities exposed to MurD inhibitors

  • Apply meta-transcriptomic approaches to identify community-wide responses

• Biogeochemical impacts: As Prochlorococcus is responsible for a significant fraction of marine primary production, perturbation of its growth through cell wall synthesis inhibition could affect:

  • Carbon fixation and sequestration rates

  • Nutrient cycling in oligotrophic waters

  • Marine food web dynamics

• Natural product discovery: The study of natural MurD inhibitors in marine environments may reveal previously uncharacterized chemical defenses and signaling molecules mediating microbial interactions in oceanic ecosystems.

These broader implications highlight the potential significance of MurD research beyond biochemical characterization to understanding fundamental ecological processes in marine systems.

What are the most promising avenues for future research on Prochlorococcus MurD?

The study of Prochlorococcus MurD offers several promising research directions:

• Structural biology advancements: Obtaining high-resolution structures of Prochlorococcus MurD in multiple conformational states would provide crucial insights into its catalytic mechanism and unique adaptations for the marine environment.

• Systems biology integration: Investigating how MurD regulation integrates with broader cellular processes in Prochlorococcus, particularly during stress responses and changing environmental conditions.

• Comparative enzymology: Systematic comparison of MurD properties across diverse marine bacteria could reveal evolutionary adaptations in cell wall biosynthesis pathways specific to different oceanic niches.

• Inhibitor development: Building on structure-based approaches to design selective inhibitors targeting Prochlorococcus MurD as research tools for studying cell wall biosynthesis in marine cyanobacteria.

• Ecological applications: Utilizing knowledge of MurD function to better understand peptidoglycan dynamics in marine microbial communities and their role in biogeochemical cycling.