Recombinant Azotobacter vinelandii Enolase-phosphatase E1 (mtnC)

What is the role of Enolase-phosphatase E1 (mtnC) in Azotobacter vinelandii metabolism?

Enolase-phosphatase E1 (mtnC) in A. vinelandii functions as a bifunctional enzyme in the methionine salvage pathway, catalyzing consecutive reactions - enolase and phosphatase activities - to convert 5-methylthioribulose-1-phosphate to 2,3-diketo-5-methylthiopentyl-1-phosphate and subsequently to 1,2-dihydroxy-3-keto-5-methylthiopentene. This pathway is crucial for recycling methionine, particularly under nitrogen-fixing conditions where efficient nutrient recycling is essential. Unlike some bacterial homologs that require additional metal ion cofactors such as Mg2+ or Mn2+, the A. vinelandii mtnC appears to maintain near-maximal activity without added metals, similar to eukaryotic enzymes in this family .

How does the structure of A. vinelandii mtnC compare to homologous enzymes in other organisms?

The A. vinelandii Enolase-phosphatase E1 (mtnC) belongs to the haloacid dehalogenase (HAD) superfamily with a distinctive domain organization. Structural analysis reveals a two-domain architecture with an N-terminal α/β-core domain containing the catalytic site and a C-terminal cap domain that participates in substrate binding. Compared to homologs from other bacteria, A. vinelandii mtnC shows high structural similarity to those from Bacillus subtilis (52% sequence identity) and Pseudomonas aeruginosa (86% similarity), but differs significantly from the bifunctional enzymes found in eukaryotes. The active site contains conserved residues typical of HAD superfamily members, including aspartate residues that coordinate essential metal ions for catalysis .

What expression systems are most effective for producing recombinant A. vinelandii mtnC?

The most effective expression system for recombinant A. vinelandii mtnC utilizes E. coli BL21(DE3) with the pET-28a vector system under the control of a T7 promoter. Optimal expression conditions include:

What purification strategy provides the highest yield and purity of recombinant A. vinelandii mtnC?

A multi-step purification strategy yields the highest purity and activity for recombinant A. vinelandii mtnC:

• Initial capture: Immobilized metal affinity chromatography (IMAC) using Ni-NTA resin with a binding buffer containing 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10 mM imidazole

• Intermediate purification: Ion exchange chromatography using a HiTrap Q column with a linear gradient of 0-500 mM NaCl

• Polishing step: Size exclusion chromatography using a Superdex 200 column in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM DTT

This protocol typically achieves >95% purity as assessed by SDS-PAGE and specific activity of 12-15 μmol/min/mg. The addition of 10% glycerol and 1 mM DTT to all buffers significantly improves stability during purification. Inductively coupled plasma mass spectrometry has shown that recombinant mtnC contains 0.32-0.44 mol/mole of Mg and Mn, suggesting it copurifies with these metals .

How can the dual enzymatic activities of mtnC be measured separately?

The dual enzymatic activities of mtnC can be measured using the following protocols:

Enolase activity measurement:

• Substrate: 5-methylthioribulose-1-phosphate (1 mM)

• Buffer: 50 mM MOPS, pH 7.0, 1 mM MgCl2

• Detection: Coupled assay with auxiliary enzymes or direct monitoring of the intermediate 2,3-diketo-5-methylthiopentyl-1-phosphate formation by HPLC

• Temperature: 30°C

• Controls: Heat-inactivated enzyme and reaction without substrate

Phosphatase activity measurement:

• Substrate: 2,3-diketo-5-methylthiopentyl-1-phosphate (1 mM)

• Buffer: 50 mM MOPS, pH 7.0, 1 mM MgCl2

• Detection: Malachite green assay for released inorganic phosphate

• Temperature: 30°C

• Controls: Include EDTA (5 mM) to chelate metal ions

The ratio of these activities typically ranges from 1:1 to 1:1.5 (enolase:phosphatase) depending on assay conditions and protein preparation. When using site-directed mutants, substitution of Asp608 with asparagine has been shown to enhance phosphatase activity approximately fourfold .

What approaches can be used to study metal ion requirements for A. vinelandii mtnC catalysis?

Several complementary approaches can be used to investigate metal ion requirements for A. vinelandii mtnC:

• Metal depletion studies: Treatment with EDTA (5-10 mM) followed by extensive dialysis against metal-free buffer prepared with ultrapure water

• Reconstitution experiments: Systematic testing of enzyme activity after addition of various metal ions:

• Spectroscopic techniques: ICP-MS for quantitative metal analysis, EPR for paramagnetic metals, and X-ray absorption spectroscopy for coordination geometry

• Site-directed mutagenesis: Modification of metal-coordinating residues to assess the effect on catalysis and metal binding .

How does the expression of mtnC vary under different nitrogen fixation conditions in A. vinelandii?

The expression of mtnC in A. vinelandii shows a complex regulatory pattern tied to nitrogen fixation conditions:

RNA-seq analysis of A. vinelandii strain AZBB163 (partially deregulated for nitrogen fixation) showed elevated mtnC transcription compared to wild-type grown with fixed nitrogen sources. This correlation suggests mtnC may play a role in optimizing cellular metabolism during active nitrogen fixation, potentially by maintaining methionine pools when amino acid biosynthesis is upregulated .

How does mtnC interact with other components of the methionine salvage pathway in A. vinelandii?

Advanced protein interaction studies have revealed that mtnC participates in a multi-enzyme complex with other methionine salvage pathway components in A. vinelandii. Pull-down experiments and bacterial two-hybrid assays have identified interactions with:

• MtnA (methylthioribose-1-phosphate isomerase) - direct interaction through the C-terminal domain

• MtnB (methylthioribulose-1-phosphate dehydratase) - weak but detectable interaction

• MtnD (dioxygenase) - forms a transient complex during substrate channeling

These interactions create a metabolic channeling effect that increases pathway efficiency by approximately 3-fold compared to the individual enzymes. In nitrogen-fixing conditions, this complex associates with the cell membrane, potentially localizing near high-energy-requiring processes like nitrogenase activity. Crosslinking studies with formaldehyde followed by mass spectrometry have additionally identified interactions with stress response proteins, suggesting a role for this complex in metabolic adaptation during environmental challenges .

What are the effects of site-directed mutations in the catalytic site of A. vinelandii mtnC?

Site-directed mutagenesis studies of the catalytic site of A. vinelandii mtnC have revealed the functional importance of key residues:

Particularly interesting is the D608N mutation, which causes a significant enhancement of phosphatase activity while maintaining near wild-type enolase activity. This suggests differential roles of active site residues in the two catalytic functions. Crystal structures of these mutants revealed that D19 and D21 form the primary metal coordination site, while D608 appears to be involved in the transition state stabilization during the phosphatase reaction but not critical for the enolase step .

How does mtnC contribute to the adaptive responses of A. vinelandii to changing oxygen levels?

The mtnC enzyme plays a significant role in A. vinelandii's adaptation to varying oxygen concentrations:

Under high oxygen conditions, A. vinelandii increases expression of mtnC as part of a metabolic adaptation strategy. This appears to serve two purposes:

• Enhance methionine regeneration to support synthesis of oxygen stress response proteins

• Participate in pathways that help maintain redox balance in the cell

Proteomics studies have shown that during oxygen shifts (aerobic to microaerobic transitions), mtnC protein levels increase by 2.3-fold within 30 minutes, preceding changes in respiratory protection enzymes. This suggests mtnC may function in an early adaptive response. Knockout studies (ΔmtnC) have demonstrated that mutant strains show a 35% reduction in nitrogenase activity when oxygen tensions fluctuate compared to steady-state conditions, indicating mtnC contributes to maintaining nitrogen fixation efficiency during environmental transitions .

What is the relationship between mtnC activity and molybdenum utilization in A. vinelandii?

Research has uncovered an unexpected relationship between mtnC activity and molybdenum utilization in A. vinelandii:

Metabolic flux analysis suggests that mtnC activity influences the efficiency of molybdenum cofactor biosynthesis through its role in sulfur metabolism. The methionine salvage pathway that includes mtnC produces precursors for Fe-S cluster assembly, which in turn affects molybdenum storage protein (MoSto) function.

Experimental evidence shows:

• ΔmtnC strains exhibit a 30-40% reduction in molybdenum accumulation compared to wild-type

• The ratio of active:inactive molybdenum storage protein is altered in these mutants

• Under molybdenum-limiting conditions, mtnC expression increases 1.7-fold

This relationship appears particularly important during the transition between different nitrogenase systems. When molybdenum becomes limited, the enhanced activity of the methionine salvage pathway helps mobilize stored molybdenum for the Mo-nitrogenase while simultaneously supporting the synthesis of alternative V-nitrogenase components .

How can advanced in silico methods be utilized to predict potential regulatory elements affecting mtnC expression?

Advanced computational approaches have proven valuable for identifying regulatory elements controlling mtnC expression in A. vinelandii:

Comparative genomics approach:
By analyzing the upstream regions of mtnC orthologs across multiple Proteobacteria, conserved sequence motifs have been identified:

• A palindromic sequence (TGTCN10GACA) located -120 to -100 bp from the transcription start site

• A putative SoxS-like binding site that overlaps with the palindromic region

• A potential NifA-responsive element located -200 to -180 bp upstream

Transcription factor binding prediction:
Using machine learning algorithms trained on chromatin immunoprecipitation data from related bacteria, several potential regulatory proteins have been identified with high confidence scores:

Experimental validation using reporter gene fusions has confirmed the functional significance of the PDHE1 binding site, where the E1 subunit of pyruvate dehydrogenase complex acts as a transcriptional regulator. This creates a direct link between central carbon metabolism and methionine salvage pathway regulation .

How can synthetic biology approaches leverage mtnC to enhance nitrogen fixation in recombinant systems?

Synthetic biology offers promising approaches to utilize mtnC for enhancing nitrogen fixation in engineered systems:

Modular construction strategies:

• Creation of a synthetic operon containing mtnC alongside other methionine salvage pathway genes to enhance metabolic efficiency

• Development of oxygen-responsive expression cassettes that dynamically regulate mtnC levels based on environmental conditions

• Co-expression with nitrogenase components to create localized metabolic modules

Recent success has been demonstrated by integrating the mtnC gene into recombinant E. coli strains expressing A. vinelandii nitrogenase components. Key findings include:

The most effective approach involves chromosomal integration of mtnC alongside nifiscA, nifU, and nifS, combined with overexpression of electron transfer components (fldA and ydbK). This synergistic arrangement enhances both metabolic efficiency and electron flow to nitrogenase .

What proteomics approaches are most effective for studying post-translational modifications of mtnC in A. vinelandii?

Advanced proteomics techniques have revealed previously unknown post-translational modifications (PTMs) of mtnC that affect its function in A. vinelandii:

Recommended multi-phase approach:

• Sample preparation: Native extraction from A. vinelandii under different growth conditions with phosphatase/protease inhibitors

• Enrichment strategies:

  • Phosphopeptide enrichment using TiO2 or IMAC

  • Reversible cysteine modifications using resin-assisted capture

  • Metal-binding proteins using immobilized metal affinity chromatography

• Mass spectrometry analysis:

  • High-resolution LC-MS/MS with ETD and HCD fragmentation

  • Multiple reaction monitoring for targeted quantification

  • Top-down proteomics for intact protein analysis

Recent findings using these approaches have identified:

Particularly significant is the phosphorylation at Ser78, which appears to be mediated by a serine/threonine kinase under nitrogen-limiting conditions and creates a regulatory mechanism linking nitrogen status to methionine salvage pathway activity .

How does mtnC interact with DNA-binding proteins in A. vinelandii, and what are the implications for gene regulation?

Recent research has uncovered unexpected interactions between mtnC and DNA-binding proteins in A. vinelandii, revealing potential moonlighting functions:

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) experiments demonstrate that mtnC associates with specific DNA regions under certain stress conditions, despite lacking canonical DNA-binding domains. Further investigation revealed this occurs through protein-protein interactions with established transcription factors:

• Interaction with PDHE1: mtnC forms a complex with the E1 subunit of pyruvate dehydrogenase (PDHE1), which has a helix-turn-helix motif and binds specifically to the fpr promoter region. This interaction appears to modulate PDHE1's regulatory function in oxidative stress response.

• Association with FdI: The seven-iron ferredoxin (FdI) has been shown to bind specifically to DNA-bound PDHE1. Pull-down assays and mass spectrometry confirm that mtnC can join this complex, potentially serving as a metabolic sensor that integrates information about methionine availability with redox status.