Recombinant Acinetobacter sp. Nicotinate phosphoribosyltransferase (pncB)

What is Acinetobacter sp. Nicotinate Phosphoribosyltransferase (pncB) and what role does it play in bacterial metabolism?

Nicotinate phosphoribosyltransferase (pncB) is a key enzyme in the NAD biosynthetic pathway of Acinetobacter species. It catalyzes the conversion of nicotinic acid (NA) to nicotinic acid mononucleotide (NaMN) in the presence of phosphoribosyl pyrophosphate (PRPP) and ATP. This reaction represents a critical step in the nicotinate salvage pathway for NAD biosynthesis .

In the broader context of bacterial metabolism, pncB functions as part of the salvage routes leading to NaMN synthesis, which eventually converges with the de novo pathway at the downstream steps catalyzed by nicotinate mononucleotide adenylyltransferase (NaMNAT) and NAD synthetase . This metabolic versatility allows Acinetobacter species to maintain NAD homeostasis under varying environmental conditions.

How does Acinetobacter NAD metabolism differ from other bacterial species?

Acinetobacter species exhibit unique features in their NAD metabolism compared to other bacteria:

• Absence of NadD: Genomes of analyzed Acinetobacter species do not encode NadD, which is replaced functionally by its distant homolog NadM .

• Dual-specificity NadM: The NadM enzyme in Acinetobacter possesses dual substrate specificity toward both nicotinate and nicotinamide mononucleotide substrates, supporting its essential role in all three routes of NAD biogenesis: de novo synthesis and both salvage pathways .

• Alternative salvage pathways: Acinetobacter possesses both deamidating and non-deamidating routes for nicotinamide salvage/recycling with distinct physiological roles .

• Transcriptional regulation: The non-deamidating route is transcriptionally regulated by an ADP-ribose-responsive repressor NrtR, providing an additional layer of metabolic control .

These differences highlight the evolutionary adaptations in Acinetobacter NAD metabolism and suggest potential targets for antimicrobial development.

What are the recommended protocols for cloning and expressing recombinant Acinetobacter pncB?

Based on successful experimental approaches, the following protocol is recommended for cloning and expressing recombinant Acinetobacter pncB:

• Gene Amplification and Cloning:

  • Amplify the pncB gene from Acinetobacter genomic DNA using PCR

  • Clone into an expression vector containing the T7 promoter (e.g., pET28a)

  • Include restriction sites (NheI and EcoRI recommended) for directional cloning

  • Add an N-terminal His6-tag for purification purposes

• Expression Conditions:

  • Transform into an E. coli expression strain (BL21(DE3) or similar)

  • Grow cultures at 37°C until mid-log phase

  • Induce protein expression with IPTG (typical concentration: 0.2-1.0 mM)

  • Continue growth at lower temperature (16-30°C) for 4-16 hours to enhance soluble protein production

• Protein Extraction and Purification:

  • Harvest cells by centrifugation

  • Lyse cells using sonication or mechanical disruption in an appropriate buffer

  • Purify using Ni-NTA affinity chromatography

  • Consider including a second purification step (ion exchange or size exclusion) for higher purity

This protocol has been successfully implemented for producing functional recombinant A. baylyi pncB protein for enzymatic studies .

How can the enzymatic activity of recombinant pncB be measured accurately?

Accurate measurement of pncB enzymatic activity can be achieved using an HPLC-based end-point assay:

• Reaction Setup:

  • Prepare reaction mixtures containing 50 mM Hepes (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 2 mM ATP, 5 mM PRPP, and 0.5 mM nicotinic acid

  • Add purified recombinant pncB enzyme (typically 1-2 nM for standard activity)

  • Incubate for 20 minutes at 30°C

• Analysis Method:

  • Analyze reaction products using ion-paired analytical C18 HPLC column (3 μm, 4.6 × 150 mm)

  • Directly quantify the NaMN product formed

  • For initial rate measurements, vary nicotinic acid concentration between 0.1–300 μM

• Specificity Testing:

  • To test specificity for alternative substrates like nicotinamide, use higher enzyme concentrations (25 μM) and substrate concentrations (0.03–20 mM)

This methodology enables precise quantification of pncB activity and determination of key kinetic parameters.

What are the critical factors affecting stability and activity of purified recombinant pncB?

Several factors significantly impact the stability and activity of purified recombinant pncB:

• Storage Conditions:

  • Temperature: Store at -80°C for long-term stability; avoid repeated freeze-thaw cycles

  • Buffer composition: Include glycerol (10-20%) and reducing agent (1-5 mM DTT or β-mercaptoethanol)

  • Protein concentration: Higher concentrations (>1 mg/ml) generally improve stability

• Reaction Conditions Affecting Activity:

  • pH: Optimal activity typically observed in the range of pH 7.0-7.5

  • Divalent cations: Mg²⁺ is essential for activity (10 mM optimal)

  • Reducing environment: DTT (1 mM) maintains reduced state of cysteine residues

  • Temperature: Activity optimum around 30°C, with decreased stability above 37°C

• Substrate Availability:

  • Both substrates (nicotinic acid and PRPP) must be present at sufficient concentrations

  • ATP is required as a cofactor for the reaction

• Inhibitory Factors:

  • High salt concentrations (>300 mM NaCl) may interfere with substrate binding

  • Product inhibition by NaMN can occur at high substrate conversion rates

  • Metal chelators (EDTA) will inhibit activity by removing essential Mg²⁺

Maintaining these critical factors within optimal ranges ensures maximal enzyme performance in experimental settings.

How does substrate specificity differ between Acinetobacter pncB and similar enzymes from other bacterial species?

Acinetobacter pncB exhibits distinct substrate specificity profiles compared to related enzymes from other bacterial species:

• Substrate Preference:

  • Acinetobacter pncB strongly prefers nicotinic acid as substrate, with significantly higher catalytic efficiency compared to nicotinamide

  • When tested for nicotinamide substrate specificity, even at high enzyme (25 μM) and substrate (0.03–20 mM) concentrations, pncB shows minimal activity

• Comparative Analysis:

  • Unlike some bacterial PncB enzymes that can process both nicotinic acid and nicotinamide with varying efficiencies, Acinetobacter pncB demonstrates strict specificity for nicotinic acid

  • This contrasts with the more promiscuous substrate utilization observed in some PncB enzymes from other bacterial species

• Structural Basis:

  • Structural differences in the substrate binding pocket likely account for the strict substrate specificity

  • Comparison of crystal structures between human NAPRT and bacterial orthologs (e.g., from E. faecalis) reveals potential molecular determinants responsible for substrate specificity differences

This strict substrate specificity has important implications for understanding the physiological role of pncB in Acinetobacter NAD metabolism and suggests that nicotinamide salvage in Acinetobacter relies primarily on alternative enzymes like NadV (nicotinamide phosphoribosyltransferase).

What is the relationship between pncB activity and bacterial pathogenicity or antibiotic resistance?

The relationship between pncB activity and bacterial pathogenicity/antibiotic resistance in Acinetobacter species reveals several important connections:

• NAD Metabolism and Pathogenicity:

  • NAD is essential for bacterial viability and virulence factor production

  • Disruption of NAD biosynthesis pathways, including those involving pncB, can attenuate bacterial virulence

  • Unlike extracellular NAPRT (eNAPRT) in humans, which mediates inflammatory responses via TLR4 activation, bacterial NAPRT (including Acinetobacter pncB) does not appear to activate inflammatory signaling

• Antibiotic Resistance Connections:

  • Carbapenem-resistant Acinetobacter baumannii (CRAb) represents a significant clinical threat, and understanding metabolic vulnerabilities is crucial for developing new therapeutics

  • NAD biosynthesis enzymes like NadD and NadE are increasingly recognized as promising drug targets

  • In Acinetobacter, the NadM enzyme (which functionally replaces NadD) has been identified as a particularly promising drug target due to its essential role in all NAD biosynthesis routes and distant homology to human counterparts

• Targeting NAD Metabolism for Antimicrobial Development:

  • While pncB itself may not be essential due to metabolic redundancy in NAD biosynthesis pathways, downstream enzymes like NadM are potential targets

  • The experimentally confirmed unconditional essentiality of nadM in Acinetobacter supports its selection as a drug target

  • In contrast, nadE appears conditionally essential, depending on specific growth conditions

Understanding these relationships provides valuable insights for developing novel therapeutic strategies against Acinetobacter infections, particularly in the context of increasing antibiotic resistance.

What methods are most effective for studying the regulation of pncB expression and activity in Acinetobacter species?

Effective methods for studying pncB regulation in Acinetobacter encompass various molecular and biochemical approaches:

• Transcriptional Regulation Analysis:

  • Electrophoretic Mobility Shift Assay (EMSA): This technique effectively identifies protein-DNA interactions involved in transcriptional regulation. For example, binding of the ADP-ribose-responsive repressor NrtR to DNA can be studied using biotinylated DNA probes containing the promoter region of interest

  • Competitive EMSA: Using a 100-fold molar excess of non-biotinylated DNA as a specific competitor helps validate binding specificity

  • Metabolite-Mediated Regulation: Pre-incubating regulatory proteins with potential effector molecules (like ADP-ribose) before adding DNA probes can reveal metabolite-dependent regulation mechanisms

• Gene Expression Analysis:

  • RNA Sequencing (RNA-seq): Provides comprehensive transcriptome analysis to identify genes co-regulated with pncB under different conditions

  • Quantitative PCR (qPCR): For targeted analysis of pncB expression levels in response to different stimuli

  • Reporter Gene Assays: Fusion of pncB promoter with reporter genes (e.g., lacZ, GFP) to monitor expression in vivo

• Genetic Manipulation Approaches:

  • Gene Knockout/Knockdown: Deletion or silencing of regulatory genes to assess their impact on pncB expression

  • Site-Directed Mutagenesis: Modification of regulatory sequences to evaluate their functional importance

  • CRISPR-Cas9 Techniques: For precise genome editing to study regulatory mechanisms

• Protein-Level Analysis:

  • Western Blotting: To quantify pncB protein levels under different conditions

  • Pulse-Chase Experiments: To measure protein turnover rates

  • Post-Translational Modification Analysis: Using mass spectrometry to identify regulatory modifications

These methodologies provide complementary approaches to understand the complex regulatory mechanisms controlling pncB expression and activity in Acinetobacter, with implications for metabolic adaptation and potential antimicrobial targeting strategies.

How do metabolic fluxes through pncB-dependent pathways differ between normal growth and stress conditions in Acinetobacter?

Metabolic flux through pncB-dependent pathways in Acinetobacter undergoes significant remodeling under stress conditions compared to normal growth:

• Normal Growth Conditions:

  • Under optimal growth conditions, Acinetobacter primarily utilizes the de novo NAD biosynthesis pathway

  • pncB activity supports nicotinate salvage as a supplementary pathway

  • Flux distribution between pathways is balanced according to substrate availability and energy requirements

  • NaMN produced by pncB enters the downstream pathway for conversion to NAD by NadM and NadE enzymes

• Stress Response Adaptations:

  • Nutrient Limitation: During nicotinamide/nicotinate limitation, salvage pathways including pncB-dependent routes become critical for maintaining NAD levels

  • Oxidative Stress: NAD consumption increases significantly during oxidative stress to support redox homeostasis, elevating the importance of all NAD biosynthetic pathways

  • Antibiotic Pressure: Exposure to antibiotics often increases metabolic flux through salvage pathways to compensate for stress-induced NAD depletion

• Pathway Integration and Regulation:

  • The dual substrate specificity of NadM (toward both nicotinate and nicotinamide mononucleotide) provides metabolic flexibility during stress adaptation

  • NrtR-mediated transcriptional regulation responds to changing ADP-ribose levels, adjusting gene expression of non-deamidating salvage pathway components during stress

  • Metabolic flux through pncB increases when the preferred de novo pathway is compromised

• Clinical Implications:

  • In infection contexts (e.g., sepsis), Acinetobacter must adapt its NAD metabolism to host defense mechanisms and nutrient restriction

  • This metabolic flexibility contributes to Acinetobacter's remarkable ability to survive in diverse environments, including healthcare settings where it presents significant clinical challenges

Understanding these dynamic flux changes provides insights into bacterial adaptation mechanisms and identifies potential vulnerability points for therapeutic intervention, particularly in drug-resistant Acinetobacter infections.

What are the most common technical challenges when working with recombinant Acinetobacter pncB and how can they be resolved?

Researchers frequently encounter these technical challenges when working with recombinant Acinetobacter pncB, along with proven solutions:

Implementing these solutions significantly improves experimental reproducibility and data quality when working with recombinant Acinetobacter pncB.

How can researchers distinguish between the activities of different phosphoribosyltransferases in complex Acinetobacter extracts?

Distinguishing between different phosphoribosyltransferase activities in complex Acinetobacter extracts requires careful experimental design:

• Substrate Specificity Approach:

  • pncB (nicotinate phosphoribosyltransferase) shows high specificity for nicotinic acid with minimal activity toward nicotinamide

  • NadV (nicotinamide phosphoribosyltransferase) preferentially utilizes nicotinamide as substrate

  • By comparing activities with different substrates, researchers can distinguish between these enzymes

• Selective Inhibition Strategy:

  • Design assays with specific inhibitors targeting individual enzymes

  • Include controls with purified recombinant enzymes to validate inhibitor specificity

  • Develop inhibition profiles for each enzyme to identify their contributions in mixed samples

• Biochemical Separation Techniques:

  • Use ion exchange chromatography to separate enzymes based on charge differences

  • Apply size exclusion chromatography to separate by molecular weight

  • Develop selective precipitation protocols using ammonium sulfate fractionation

• Genetic Approaches:

  • Create knockout strains lacking specific phosphoribosyltransferases

  • Compare activities in wild-type vs. knockout extracts to determine contribution of each enzyme

  • Complement knockout strains with recombinant enzymes to confirm specificity

• Analytical Method Refinement:

  • Optimize HPLC conditions to clearly separate reaction products from different phosphoribosyltransferases

  • Employ LC-MS to distinguish between products based on mass differences

  • Develop enzyme-coupled spectrophotometric assays with specificity for particular reaction products

These approaches, particularly when used in combination, enable researchers to accurately characterize the activities of different phosphoribosyltransferases in complex Acinetobacter extracts.

What are the most promising research directions for targeting Acinetobacter pncB and related NAD biosynthesis enzymes for antimicrobial development?

Several promising research directions exist for targeting Acinetobacter NAD biosynthesis enzymes:

• Rational Drug Design Targeting NadM:

  • NadM has been experimentally confirmed as unconditionally essential in Acinetobacter, making it a prime target

  • Its distant homology to human counterparts reduces potential toxicity concerns

  • Structure-based drug design leveraging unique features of bacterial NadM could yield selective inhibitors

• Combination Therapies:

  • Targeting multiple enzymes in NAD biosynthesis simultaneously may prevent metabolic bypass

  • Combining NAD biosynthesis inhibitors with existing antibiotics could enhance efficacy against resistant strains

  • Identifying synergistic combinations through high-throughput screening approaches

• Pathway-Specific Inhibitor Development:

  • Design prodrugs activated by bacterial-specific enzymes in the NAD pathway

  • Develop inhibitors that exploit the unique dual substrate specificity of Acinetobacter NadM

  • Screen for natural products that specifically target bacterial NAD biosynthesis

• Novel Delivery Strategies:

  • Engineer nanoparticle delivery systems to enhance penetration into Acinetobacter biofilms

  • Develop targeted delivery approaches to increase local concentrations of inhibitors

  • Design formulations to overcome permeability barriers in Acinetobacter

• Genomic and Metabolomic Analysis:

  • Conduct comparative genomics across clinical isolates to identify conserved targets

  • Perform metabolomic studies to identify bottlenecks in NAD metabolism during infection

  • Use systems biology approaches to model pathway vulnerabilities under different conditions

This multifaceted approach addressing both established targets (NadM, NadE) and broader pathway vulnerabilities offers promising avenues for combating increasingly resistant Acinetobacter infections, particularly carbapenem-resistant A. baumannii that poses significant clinical challenges .

How might evolutionary analysis of pncB across Acinetobacter species reveal insights into metabolic adaptation and pathogenicity?

Evolutionary analysis of pncB across Acinetobacter species provides valuable insights into bacterial adaptation and pathogenicity:

• Comparative Genomics:

  • Analysis of pncB sequence conservation across pathogenic and non-pathogenic Acinetobacter species reveals selection pressures

  • Identification of species-specific variations may correlate with ecological niches and pathogenic potential

  • Comparison with pncB from other bacterial genera highlights unique Acinetobacter adaptations

• Structural Evolution:

  • Mapping sequence variations onto protein structures can identify functionally important regions

  • Comparing structures of human NAPRT with bacterial orthologs reveals distinct features that could inform selective targeting

  • Analysis of key structural differences between Acinetobacter pncB and enzymes from other bacteria (e.g., E. faecalis) highlights evolutionary divergence

• Metabolic Network Adaptation:

  • The unique NAD metabolism in Acinetobacter, including the absence of NadD and its functional replacement by NadM, represents significant evolutionary adaptation

  • Tracing the evolution of this metabolic reconfiguration may reveal how Acinetobacter adapted to specific environmental pressures

  • Identifying co-evolving components within the NAD metabolic network provides insights into functional dependencies

• Host-Pathogen Interface:

  • Unlike human NAPRT, which can act as an extracellular signaling molecule activating inflammatory responses via TLR4, bacterial NAPRT (including from Acinetobacter) lacks this signaling function

  • This evolutionary difference has important implications for host-pathogen interactions during infection

  • Understanding how these differences evolved may reveal mechanisms by which Acinetobacter evades or modulates host immune responses

Such evolutionary analyses not only enhance our understanding of Acinetobacter biology but also identify potential vulnerabilities for therapeutic exploitation, particularly relevant given the increasing prevalence of multidrug-resistant strains in healthcare settings.