Recombinant Methylobacterium chloromethanicum Nucleoside diphosphate kinase (ndk)

What is the fundamental biological function of Nucleoside Diphosphate Kinase (NDK) in Methylobacterium chloromethanicum?

NDK catalyzes the reversible exchange of the γ-phosphate between nucleoside triphosphate (NTP) and nucleoside diphosphate (NDP), playing a critical role in nucleotide metabolism. Beyond this canonical phosphotransferase activity, NDK likely has additional functions in M. chloromethanicum's cellular processes similar to other bacterial NDKs. The enzyme maintains nucleotide pool balance by transferring phosphate groups between different nucleotides, which is essential for DNA synthesis, cell division, and other metabolic processes .

How does M. chloromethanicum NDK compare structurally to NDKs from other organisms?

While specific structural data for M. chloromethanicum NDK is not fully characterized, NDK proteins typically share common structural elements including a βαββαβ or "ferredoxin" folding pattern. Based on structural conservation among bacterial NDKs, M. chloromethanicum NDK likely forms oligomeric structures, possibly as dimers, tetramers, or hexamers, similar to NDKs from other organisms. This oligomerization suggests that M. chloromethanicum NDK can interact with other molecules to perform its biological functions .

What role might NDK play in M. chloromethanicum's unique ability to utilize chloromethane?

M. chloromethanicum is distinguished by its ability to grow using chloromethane as its sole carbon and energy source. While NDK is not directly involved in the primary chloromethane utilization pathway (which relies on CmuA and CmuB enzymes), it may play a supporting role in energy metabolism by maintaining nucleotide homeostasis during growth on this unusual carbon source. The chloromethane utilization pathway involves tetrahydrofolate-dependent enzymes, and NDK activity may indirectly support this metabolic process by ensuring adequate supply of nucleotides for cellular functions .

What expression systems are most effective for producing recombinant M. chloromethanicum NDK?

For recombinant expression of M. chloromethanicum NDK, Escherichia coli-based systems are most commonly used due to their high yield, cost-effectiveness, and rapid growth. The gene encoding NDK can be cloned into expression vectors containing appropriate promoters (such as T7) and affinity tags (like hexahistidine) to facilitate purification. When optimizing expression conditions, researchers should consider parameters such as growth temperature (typically 16-30°C), IPTG concentration for induction (0.1-1.0 mM), and post-induction time (4-18 hours) .

What purification strategy yields the highest purity and activity for recombinant M. chloromethanicum NDK?

A multi-step purification approach typically yields the best results:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin to capture His-tagged NDK

• Intermediate purification: Ion exchange chromatography (typically anion exchange)

• Polishing step: Size exclusion chromatography to separate oligomeric forms

This strategy can yield recombinant NDK with >90% purity suitable for enzymatic assays, structural studies, and biochemical characterization. Buffer conditions should be optimized to maintain enzyme stability, typically including:

Optimization of these conditions may be necessary depending on the specific properties of M. chloromethanicum NDK .

How can the phosphate transferase activity of M. chloromethanicum NDK be measured accurately?

The phosphate transferase activity of M. chloromethanicum NDK can be measured using a coupled enzyme assay system similar to that used for other NDKs. The standard method involves:

• Coupling NDK reaction with pyruvate kinase and lactate dehydrogenase enzymes

• Monitoring NADH oxidation spectrophotometrically at 340 nm

In this assay, NDK converts ATP + dTDP to ADP + dTTP. The ADP released is then measured by the coupled enzyme system, where pyruvate kinase converts phosphoenolpyruvate and ADP to pyruvate and ATP, and lactate dehydrogenase converts pyruvate and NADH to lactate and NAD+. The decrease in NADH absorption correlates directly with NDK activity.

The reaction mixture typically contains:

• 50 mM Tris-HCl (pH 7.5)

• 5 mM MgCl₂

• 1 mM ATP

• 0.5 mM dTDP (or other NDP substrate)

• 0.2 mM NADH

• 1 mM phosphoenolpyruvate

• 2 units pyruvate kinase

• 2 units lactate dehydrogenase

• Purified recombinant NDK (1-10 μg)

How does temperature and pH affect M. chloromethanicum NDK activity, and what are the optimal conditions for enzymatic assays?

For methylotrophic bacteria like M. chloromethanicum that grow optimally at moderate temperatures, NDK activity should be assayed across these parameters:

Thermal stability studies using differential scanning fluorimetry (DSF) can also provide insights into the enzyme's stability under different buffer conditions. Since M. chloromethanicum grows at 30°C on chloromethane-containing media, its NDK likely has optimal activity near this temperature .

Which amino acid residues in M. chloromethanicum NDK are essential for catalytic activity?

Based on structure-function studies of NDKs from other organisms, several conserved residues are likely critical for M. chloromethanicum NDK activity:

• A histidine residue in the active site (equivalent to H117 in AfNDK) that serves as the phosphate acceptor during catalysis

• An arginine residue (equivalent to R104 in AfNDK) that stabilizes the nucleotide binding

• An aspartic acid residue (equivalent to D120 in AfNDK) that coordinates with the metal ion cofactor

Site-directed mutagenesis of these residues would likely produce variants with significantly decreased enzymatic activity. A systematic alanine-scanning mutagenesis approach can be used to identify all residues essential for catalysis and substrate binding .

How might the oligomeric state of M. chloromethanicum NDK affect its function in vivo?

NDK proteins from various organisms exist in different oligomeric states including dimers, tetramers, and hexamers. The oligomeric state influences:

• Stability: Higher-order oligomers often show increased thermal stability

• Substrate specificity: Different oligomeric states may favor different nucleotide substrates

• Protein-protein interactions: The quaternary structure affects interaction with partner proteins

• Subcellular localization: Oligomeric state may influence cellular distribution

To determine the native oligomeric state of M. chloromethanicum NDK, analytical techniques such as size exclusion chromatography, dynamic light scattering, and native PAGE should be employed. Crosslinking experiments and multi-angle light scattering can provide additional insights into the relationship between oligomeric state and function .

How does M. chloromethanicum NDK compare phylogenetically with NDKs from other methylotrophic bacteria?

• Multiple sequence alignment of NDK proteins from diverse methylotrophs

• Construction of phylogenetic trees using maximum likelihood or Bayesian methods

• Identification of sequence motifs unique to methylotrophic bacteria

Such comparative genomic analyses may reveal whether M. chloromethanicum NDK contains unique adaptations related to the organism's specialized metabolism, particularly its ability to utilize chloromethane as a carbon source .

Are there significant structural or functional differences between NDK from M. chloromethanicum and other bacterial NDKs such as that from Mycobacterium tuberculosis?

Mycobacterium tuberculosis NDK has been shown to interact with Rv1273c, a probable drug ABC transporter ATP-binding protein, contributing to mycobacterium virulence within macrophages. Comparative structural and functional analyses between M. chloromethanicum NDK and M. tuberculosis NDK would involve:

• Homology modeling of M. chloromethanicum NDK using crystal structures of related NDKs

• Molecular docking and simulation studies to identify potential protein-protein interactions

• Experimental validation using pull-down assays and far-western blotting to identify binding partners

These analyses could reveal whether M. chloromethanicum NDK possesses specialized protein-protein interactions related to its ecological niche and metabolic capabilities .

How can recombinant M. chloromethanicum NDK be used to study chloromethane metabolism pathways?

Recombinant M. chloromethanicum NDK can serve as a valuable tool for studying the organism's unique chloromethane metabolism through several approaches:

• Metabolic flux analysis: Using labeled nucleotides to track phosphate transfer during growth on chloromethane

• Protein-protein interaction studies: Identifying NDK interactions with components of the chloromethane utilization pathway

• In vivo localization: Using fluorescently tagged NDK to determine its cellular localization during growth on different carbon sources

• Transcriptional regulation: Analyzing NDK expression patterns in response to chloromethane and other carbon sources

While NDK is not directly involved in the chloromethane dehalogenation pathway catalyzed by CmuA and CmuB enzymes, it may play a supporting role in nucleotide metabolism during growth on this challenging carbon source .

What insights might M. chloromethanicum NDK provide about bacterial adaptation to unusual carbon sources?

M. chloromethanicum has evolved specialized pathways for utilizing chloromethane, a C1 compound that most organisms cannot metabolize. Studying its NDK in comparison with NDKs from bacteria utilizing more common carbon sources may reveal:

• Metabolic adaptations: Whether NDK activity or regulation is modified to support growth on chloromethane

• Energy conservation strategies: How nucleotide metabolism is optimized under the constraints of C1 metabolism

• Stress responses: Whether NDK plays a role in managing oxidative or other stresses during chloromethane metabolism

Gene expression studies comparing NDK transcription during growth on chloromethane versus methanol could provide valuable insights into its role in supporting specialized metabolism .

What high-resolution structural techniques would be most informative for studying M. chloromethanicum NDK?

Several complementary structural biology approaches would provide comprehensive insights:

These structural data would facilitate structure-based drug design targeting NDK and provide a foundation for understanding its catalytic mechanism in detail .

How might CRISPR-Cas9 genome editing be applied to study NDK function in M. chloromethanicum?

CRISPR-Cas9 genome editing could revolutionize the study of NDK function in M. chloromethanicum through:

• Gene knockout studies: Creating clean NDK deletion mutants to determine its essentiality and phenotypic effects

• Point mutations: Introducing specific mutations at the endogenous locus to test structure-function hypotheses

• Promoter modifications: Altering NDK expression levels to assess dosage effects

• Tagging: Adding fluorescent or affinity tags to the endogenous gene for localization and interaction studies

The phenotypic effects of these genetic modifications could be assessed during growth on different carbon sources, particularly comparing chloromethane and methanol utilization efficiency .

What bioinformatic approaches might reveal novel insights about the role of NDK in M. chloromethanicum metabolism?

Advanced bioinformatic analyses can uncover hidden relationships between NDK and other aspects of M. chloromethanicum metabolism:

• Gene co-expression network analysis: Identifying genes whose expression patterns correlate with NDK during growth on different carbon sources

• Protein-protein interaction prediction: Using computational methods to predict potential binding partners

• Metabolic modeling: Incorporating NDK into genome-scale metabolic models of M. chloromethanicum to predict the effects of altered NDK activity

• Comparative genomics: Analyzing the genomic context of NDK genes across multiple Methylobacterium species to identify conserved gene clusters

These approaches could generate hypotheses about NDK's role in the broader metabolic network, particularly in relation to the specialized chloromethane utilization pathway .