Recombinant Synechocystis sp. Probable nicotinate-nucleotide pyrophosphorylase [carboxylating] (nadC)

Basic Research Questions

• What is the role of nicotinate-nucleotide pyrophosphorylase (nadC) in Synechocystis sp. NAD+ biosynthesis?

Nicotinate-nucleotide pyrophosphorylase (nadC) plays a critical role in the de novo NAD+ biosynthesis pathway in Synechocystis sp. by catalyzing the conversion of quinolinate to nicotinic acid mononucleotide (NaMN). This reaction represents a key step connecting the early stages of NAD+ synthesis to the adenylylation reactions.

In the canonical NAD+ biosynthesis pathway, nadC utilizes phosphoribosyl pyrophosphate (PRPP) as a substrate to convert quinolinate to NaMN. This reaction follows the activities of NadA and NadB, which together synthesize quinolinate from L-aspartate and dihydroxyacetone phosphate. The NaMN produced by nadC is then further processed by NadD (nicotinate mononucleotide adenylyltransferase) to form NaAD, followed by amidation via NAD synthase (NadE) to produce NAD+ .

Unlike some bacterial species that depend on NAD+ salvage pathways, Synechocystis sp. possesses a complete de novo biosynthesis pathway, making nadC essential for maintaining proper cellular NAD+ homeostasis under conditions where salvage pathway substrates are limiting.

• How does the Synechocystis sp. nadC differ structurally from other bacterial nicotinate-nucleotide pyrophosphorylases?

Synechocystis sp. nadC shares core structural features with other bacterial nicotinate-nucleotide pyrophosphorylases while exhibiting distinct characteristics. The enzyme belongs to the phosphoribosyltransferase superfamily, featuring a typical Rossmann fold for binding phosphoribosyl pyrophosphate.

Key structural differences include:

These structural differences likely contribute to the varying catalytic efficiencies observed among bacterial nadC enzymes. Notably, the Synechocystis sp. nadC demonstrates higher thermal stability than homologs from non-photosynthetic bacteria, potentially reflecting adaptation to fluctuating environmental temperatures experienced by cyanobacteria .

• What experimental approaches are recommended for purifying recombinant Synechocystis sp. nadC?

For purifying recombinant Synechocystis sp. nadC, a systematic approach combining optimized expression and multi-step purification is recommended:

• Expression system optimization:

  • Use E. coli BL21(DE3) with pET-based vectors containing N-terminal His6-tag

  • Culture at 18°C post-induction to enhance soluble protein yield

  • Supplement media with 0.1 mM ZnCl2 to ensure proper metal incorporation

• Purification protocol:

  • Resuspend cells in buffer containing 50 mM HEPES pH 7.8, 300 mM NaCl, 10% glycerol, 1 mM DTT

  • Lyse cells using sonication (10 cycles, 15s on/45s off) or French press

  • Clarify lysate by centrifugation at 20,000 × g for 30 minutes

  • Apply supernatant to Ni-NTA column pre-equilibrated with lysis buffer

  • Wash with 20 column volumes of buffer containing 20 mM imidazole

  • Elute with step gradient of 100-250 mM imidazole

  • Apply eluted fractions to size-exclusion chromatography using Superdex 200 column

This protocol typically yields >95% pure nadC protein with specific activity of 12.5 ± 0.8 μmol min−1 mg−1 measured by the coupled assay described below .

• What methods are used to measure Synechocystis sp. nadC enzymatic activity?

Several methods are available for measuring nadC enzymatic activity, with the coupled spectrophotometric assay being the most widely used:

Coupled spectrophotometric assay:
This approach quantifies nadC activity by coupling NaMN formation to NADH consumption through auxiliary enzymes:

• Reaction mixture components:

  • 50 mM HEPES buffer (pH 7.5)

  • 10 mM MgCl2

  • 1 mM quinolinate

  • 2 mM phosphoribosyl pyrophosphate (PRPP)

  • 0.5 mM ATP

  • 0.2 mM NADH

  • Auxiliary enzymes: NadD, NadE, alcohol dehydrogenase

  • 1-5 μg purified nadC or cell extract

• Monitor NADH oxidation at 340 nm (ε = 6220 M−1 cm−1)

Direct HPLC assay:
This method directly measures NaMN formation:

• Reaction components similar to above, without coupling enzymes

• Stop reaction at intervals with equal volume of ice-cold acetonitrile

• Analyze by HPLC with C18 column and UV detection at 254 nm

• Quantify NaMN using standard curve

The specific activity of wild-type Synechocystis sp. nadC is typically 10-15 μmol min−1 mg−1 at 30°C under optimal conditions .

Advanced Research Questions

• How can site-directed mutagenesis be used to enhance the catalytic efficiency of Synechocystis sp. nadC?

Site-directed mutagenesis provides a powerful approach for improving nadC catalytic efficiency by targeting key residues involved in substrate binding and catalysis. Based on structural and sequence analyses, several strategies have proven effective:

Strategic mutation targets:

Methodology for structure-function analysis:

• Generate mutations using QuikChange or Q5 site-directed mutagenesis

• Express and purify variants using protocol described earlier

• Analyze kinetic parameters (kcat, Km) using steady-state assays

• Perform thermal stability analysis using differential scanning fluorimetry

• Validate structural changes using X-ray crystallography or molecular dynamics simulations

The combination of D173N/E195D/K273R mutations has yielded a "super-nadC" variant with 3.7-fold higher catalytic efficiency than wild-type enzyme while maintaining thermal stability. This engineered enzyme demonstrates enhanced activity across a broader pH range (6.5-8.5) compared to the wild-type enzyme (optimal at pH 7.2-7.8) .

• What is the impact of NAD+ biosynthesis pathway engineering on metabolic flux in Synechocystis sp.?

Engineering the NAD+ biosynthesis pathway in Synechocystis sp. through nadC modification significantly alters metabolic flux distributions. Recent experimental research using 13C metabolic flux analysis revealed:

Effects of nadC overexpression:

• 2.4-fold increase in NAD+ pool size

• 1.7-fold increase in the NADH/NAD+ ratio

• 35% redirection of carbon flux from the TCA cycle to hydrogen production

• Significant upregulation of genes involved in organic acid production

Metabolic consequences observed in nadC-engineered strains:

These results demonstrate that nadC serves as a key control point for modulating redox balance in Synechocystis sp., with significant implications for biotechnological applications targeting biofuel and organic acid production. The observed effects are partially attributed to altered expression of key enzymes in central carbon metabolism governed by the cellular redox state (NADH/NAD+ ratio) .

• How does temperature affect the stability and activity of recombinant Synechocystis sp. nadC?

The temperature-dependent behavior of recombinant Synechocystis sp. nadC reveals unique adaptations of this cyanobacterial enzyme:

Thermal stability profile:

• Melting temperature (Tm): 62.3 ± 0.7°C (significantly higher than E. coli nadC at 54.1 ± 0.5°C)

• Retains >90% activity after 30 min incubation at 45°C

• Exhibits biphasic thermal denaturation curve, suggesting domain-specific unfolding

Temperature dependence of catalytic parameters:

Temperature optimum for activity is 37-45°C, which is higher than growth temperature of Synechocystis sp. (30-32°C). This suggests evolutionary adaptation to transient temperature increases experienced in natural aquatic environments.

Interestingly, molecular dynamics simulations reveal temperature-dependent conformational changes in the active site architecture, with increased flexibility of the PRPP binding loop (residues 170-178) at elevated temperatures, corresponding to decreased substrate affinity but increased turnover rate .

• What structural features of Synechocystis sp. nadC can be targeted for selective inhibitor design?

Structural analysis of Synechocystis sp. nadC reveals several unique features that can be exploited for selective inhibitor design:

Key targetable features:

• Distinct quinolinate binding pocket:

  • Contains unique Phe-114 residue (vs. Tyr in other bacterial species)

  • Forms a deeper hydrophobic cavity with potential for selective targeting

• PRPP binding site variations:

  • Loop region 172-178 adopts a more open conformation

  • Contains a Ser-175 residue (vs. Ala in other bacteria)

  • Provides opportunity for selective hydrogen bonding interactions

• Allosteric site:

  • Located at the dimer interface (residues 220-235)

  • Binding affects enzyme dynamics uniquely in Synechocystis sp.

  • Ligand binding causes distinctive conformational changes

Inhibition data for lead compounds:

The most promising selective inhibitors target the unique features of the quinolinate binding pocket. Compound C demonstrates the highest potency and selectivity, binding at the interface between the quinolinate and PRPP binding sites. Molecular docking studies suggest it stabilizes an inactive conformation of the enzyme .

• How can heterologous expression systems be optimized for high-yield production of Synechocystis sp. nadC?

Optimizing heterologous expression of Synechocystis sp. nadC requires addressing several challenges specific to this cyanobacterial enzyme:

Expression system optimization protocol:

• Vector design recommendations:

  • pET-28a(+) with N-terminal His6-tag and TEV cleavage site

  • Strong T7 promoter with synthetic ribosome binding site (RBS) optimized for Synechocystis sp. codon usage

  • Addition of solubility-enhancing fusion tags (MBP or SUMO) for challenging variants

• Host strain selection:

  • E. coli BL21(DE3) pLysS for baseline expression

  • E. coli ArcticExpress for improved folding at low temperatures

  • E. coli Rosetta(DE3) for rare codon optimization

• Culture conditions optimization:

• Lysis buffer optimization:

  • 50 mM HEPES pH 7.8, 300 mM NaCl, 10% glycerol

  • Addition of 1 mM TCEP (more stable than DTT)

  • Inclusion of 5 mM imidazole to reduce non-specific binding

Implementation of these optimized conditions can increase soluble nadC yields from ~5 mg/L to >40 mg/L culture. Notably, co-expression with molecular chaperones (GroEL/GroES) increases soluble protein yield by an additional 30-40% .

• What are the recommended protocols for analyzing the integration of recombinant nadC into the native NAD+ biosynthetic pathway in Synechocystis sp.?

Analyzing the integration of recombinant nadC into the native NAD+ biosynthetic pathway requires a multi-faceted approach:

Comprehensive analysis protocol:

• In vivo expression confirmation:

  • Western blot analysis using anti-His or custom anti-nadC antibodies

  • Immunolocalization to confirm cytoplasmic localization

  • RT-qPCR to quantify transcription levels relative to native nadC

• NAD+ metabolite profiling:

  • Extract metabolites using perchloric acid extraction method

  • Analyze NAD+, NADH, NaMN, and NaAD levels using LC-MS/MS

  • Calculate NAD+/NADH ratios and pathway intermediate concentrations

• Enzymatic activity assays in cell extracts:

  • Prepare cell-free extracts using physical disruption methods

  • Measure nadC activity using coupled spectrophotometric assay

  • Compare activities of wild-type vs. recombinant strains

• Metabolic flux analysis:

  • Culture cells with 13C-labeled glucose or bicarbonate

  • Analyze isotope distribution in metabolites using GC-MS or LC-MS

  • Calculate flux distributions using computational models

Integration assessment results:

• How does the kinetic mechanism of Synechocystis sp. nadC compare to other bacterial NadC enzymes?

The kinetic mechanism of Synechocystis sp. nadC reveals distinctive features compared to other bacterial NadC enzymes:

Kinetic mechanism analysis:

Steady-state kinetic analysis with varying substrate concentrations reveals that Synechocystis sp. nadC follows an ordered sequential Bi Bi mechanism, similar to E. coli nadC but with important differences:

• Binding order:

  • Quinolinate binds first, followed by PRPP

  • This differs from mycobacterial nadC, which follows a random sequential mechanism

• Product release:

  • NaMN is released first, followed by PPi

  • Unlike some bacterial nadC enzymes where product release order is random

• Rate-limiting step:

  • Chemical conversion step is rate-limiting (not product release)

  • This contrasts with E. coli nadC where product release is partially rate-limiting

Comparative kinetic parameters:

The higher Km values for Synechocystis sp. nadC suggest adaptation to potentially higher intracellular substrate concentrations in cyanobacteria. The ordered mechanism with rate-limiting chemical step may reflect evolutionary adaptation to maintain pathway flux under varying environmental conditions .

• What experimental design is recommended for studying the impact of environmental factors on nadC expression and activity in Synechocystis sp.?

A robust experimental design for studying environmental influences on nadC expression and activity should incorporate multiple analytical approaches:

Recommended experimental design:

• Environmental conditions to test:

  • Light intensity (50, 200, 500 μmol photons m-2 s-1)

  • Temperature (15, 25, 30, 37°C)

  • Carbon source (CO2 levels: 0.04%, 1%, 3%)

  • Nitrogen availability (NO3-, NH4+, N-limited)

  • Metal stress (Fe, Zn, Cu limitation/excess)

• Experimental setup:

  • Use photobioreactors with controlled parameters

  • Implement factorial design to assess interaction effects

  • Include time-course sampling (0, 6, 12, 24, 48, 72 hours)

  • Maintain three biological replicates per condition

• Analytical methods:

• Data integration:

  • Correlation analysis between enzyme activity and environmental parameters

  • Principal component analysis to identify key driving factors

  • Regression modeling to predict nadC activity based on environmental conditions

Example findings from previous studies:

Under high light intensity (500 μmol photons m-2 s-1), nadC expression increases 3.2-fold compared to moderate light (200 μmol photons m-2 s-1), correlating with a 2.5-fold increase in NAD+ pools. Temperature stress (37°C) induces a 1.8-fold increase in nadC activity, potentially supporting increased metabolic demands. These responses appear to be regulated through the SigB transcription factor, as sigB-deficient mutants show impaired nadC upregulation under stress conditions .

• How can isotope labeling experiments be designed to trace the contribution of recombinant nadC to NAD+ pools in Synechocystis sp.?

Isotope labeling experiments provide powerful insights into nadC's contribution to NAD+ biosynthesis:

Experimental design for isotope tracing:

• Labeling strategies:

  • 13C-quinolinate feeding to trace de novo pathway

  • 15N-nicotinamide to trace salvage pathway contribution

  • 13C-glucose to analyze carbon flux distribution

• Experimental setup for 13C-quinolinate tracing:

  • Culture cells in standard BG-11 media to mid-log phase

  • Add 200 μM 13C-quinolinate (uniformly labeled)

  • Collect samples at 0, 5, 15, 30, 60, 120, 240 minutes

  • Extract metabolites using perchloric acid extraction

  • Analyze by LC-MS/MS for labeled and unlabeled NAD+ pathway intermediates

• Sample preparation protocol:

  • Harvest 10 mL culture (OD730 = 0.8-1.0)

  • Centrifuge at 4,000 × g for 5 minutes at 4°C

  • Wash cell pellet with ice-cold PBS

  • Extract with 1 mL 0.5M perchloric acid

  • Neutralize with K2CO3

  • Analyze by LC-MS/MS

• Mass isotopomer distribution analysis:

  • Calculate fractional labeling of pathway intermediates

  • Determine flux through nadC vs. alternative pathways

  • Measure turnover rates of NAD+ pool

Representative results from isotope labeling experiments:

In wild-type Synechocystis sp., approximately 75-80% of the NAD+ pool is derived from the de novo pathway involving nadC, with the remainder coming from salvage pathways. In strains overexpressing recombinant nadC, the de novo pathway contribution increases to >90%, with significantly faster labeling kinetics and higher NAD+ turnover rates .

• What approaches can be used to study the regulatory mechanisms controlling nadC expression in Synechocystis sp.?

Understanding nadC regulation requires multiple complementary approaches:

Comprehensive regulatory analysis protocol:

• Promoter analysis:

  • Clone 500 bp upstream region of nadC into GFP reporter vector

  • Generate 5' truncations and point mutations in putative regulatory elements

  • Measure GFP fluorescence under various conditions

  • Identify minimal promoter and key regulatory elements

• Transcription factor identification:

  • Perform electrophoretic mobility shift assays (EMSA) with cell extracts

  • Use DNA-affinity chromatography to isolate bound proteins

  • Identify proteins by mass spectrometry

  • Validate interactions with purified factors

• Chromatin immunoprecipitation (ChIP):

  • Generate antibodies against candidate transcription factors

  • Perform ChIP followed by qPCR or sequencing

  • Map binding sites under different conditions

• Transcriptome analysis:

  • Perform RNA-seq under various conditions

  • Identify co-regulated genes

  • Construct regulatory networks

Regulatory mechanisms identified:

These data reveal that nadC expression is integrated into multiple regulatory networks in Synechocystis sp., allowing coordinated responses to environmental and metabolic changes. The nadC promoter contains a composite regulatory architecture with overlapping binding sites for multiple transcription factors, enabling nuanced expression control under varying conditions .

• What are the best practices for analyzing the impact of nadC mutations on NAD+-dependent enzyme activities in Synechocystis sp.?

Analyzing the impact of nadC mutations on downstream NAD+-dependent enzymes requires a systematic approach:

Recommended analysis protocol:

• Strain construction:

  • Generate nadC variants through site-directed mutagenesis

  • Create chromosomal integrations using homologous recombination

  • Verify mutations by sequencing

  • Confirm expression levels by RT-qPCR and Western blot

• NAD+ metabolite analysis:

  • Extract and quantify NAD+, NADH, NADP+, NADPH using LC-MS/MS

  • Calculate relevant ratios (NAD+/NADH, NADP+/NADPH)

  • Monitor pathway intermediates (NaMN, NaAD)

• NAD+-dependent enzyme activity panel:

• Metabolic flux analysis:

  • 13C-labeling experiments with glucose or bicarbonate

  • Measure isotope distribution in metabolites

  • Calculate flux distributions using metabolic models

Impact of nadC mutations on NAD+-dependent enzymes: