Recombinant Acinetobacter sp. Type III pantothenate kinase (coaX)

Basic Research Questions

• What is Type III pantothenate kinase (CoaX) and why is it significant in Acinetobacter research?

  Type III pantothenate kinase (PanK), encoded by the coaX gene in Acinetobacter species, catalyzes the first and rate-limiting step in the essential coenzyme A (CoA) biosynthesis pathway. This enzyme phosphorylates pantothenate (vitamin B5) to 4′-phosphopantothenate, initiating the CoA synthesis cascade. The significance of Type III PanK in Acinetobacter research stems from several key factors:

  • It is essential for bacterial viability as demonstrated in conditional mutant studies

  • It exhibits low sequence identity (approximately 28%) with other corresponding enzymes

  • Its structure is significantly different from human PanK, making it an attractive antimicrobial drug target

  • Unlike Type I PanKs, Type III enzymes are not subject to feedback inhibition by CoASH and do not recognize N-pentylpantothenamide

  Understanding AbPanK structure and function provides critical insights for developing targeted antimicrobials against multidrug-resistant Acinetobacter baumannii, a serious hospital-acquired pathogen.

• How does Acinetobacter Type III PanK differ structurally from other PanK types?

  Acinetobacter Type III PanK belongs to the evolutionarily distinct Type III family of pantothenate kinases, which differs significantly from Type I (bacterial) and Type II (eukaryotic) enzymes:

  • Type III PanKs show very limited sequence identity with Type I and Type II enzymes

  • Crystal structures reveal unique binding modes for both pantothenate and ATP substrates

  • Type III PanKs typically crystallize in P2 space group with characteristic cell dimensions (for AbPanK: a=165 Å, b=260 Å, c=197 Å and α=90.0, β=113.60, γ=90.0)

  • Type III enzymes lack the regulatory domains present in Type I PanKs that enable feedback inhibition by CoASH

  • The binding pocket for pantothenate in Type III PanKs contains distinctive motifs compared to other types

  These structural differences enable selective targeting of bacterial Type III PanKs without affecting human Type II enzymes, which is crucial for antimicrobial development.

Advanced Research Questions

• What are the kinetic parameters and substrate binding characteristics of Acinetobacter Type III PanK?

  Biochemical characterization studies of AbPanK have revealed detailed kinetic and binding properties:

  Parameter	Value	Technique
  Pantothenate binding affinity (Kd)	1.2 × 10⁻⁸ M	Spectrofluorometry
  ATP binding affinity (Kd)	3.7 × 10⁻³ M	Spectrofluorometry
  Hydrodynamic radius	Corresponds to MW of 29.55 kDa	Dynamic light scattering
  Oligomeric state	Homogeneous solution	Dynamic light scattering
  Space group	P2	X-ray crystallography

  AbPanK demonstrates strong affinity for pantothenate and moderate affinity for ATP, with binding constants validated through both spectrofluorometric analyses and kinase assay determination of Km values . The strong pantothenate binding coupled with moderate ATP affinity suggests a sequential binding mechanism where pantothenate binding likely precedes ATP binding. This differs from some Type I PanKs and provides insights into the catalytic mechanism that can be exploited for inhibitor design.

• How can structural knowledge of Type III PanK be leveraged for rational drug design?

  Structural insights from Type III PanK studies offer several approaches for rational drug design:

  • Target the unique pantothenate binding pocket that differs from human Type II PanK

  • Exploit differences in ATP binding modes between bacterial Type III and human Type II enzymes

  • Design competitive inhibitors that mimic substrate binding but prevent catalysis

  • Focus on bacterial-specific structural elements absent in human PanKs

  • Utilize crystal structures of PanK complexed with substrates (pantothenate, ATP) and products (4′-phosphopantothenate) to identify critical interaction points

  The availability of crystal structures for Type III PanKs from multiple organisms (A. baumannii, P. aeruginosa, T. maritima) provides comparative structural data that can identify conserved catalytic features among bacterial PanKs while highlighting differences from human enzymes . Effective inhibitors should ideally target bacterial-specific features while avoiding interactions with structural elements shared with human PanKs.

• What is the relationship between Type III PanK and other metabolic pathways in Acinetobacter?

  Type III PanK interfaces with several metabolic networks in Acinetobacter beyond just CoA biosynthesis:

  • In Bacillus anthracis (which has a similar Type III PanK), the coaX gene is part of a tricistronic operon with hslO (encoding heat shock protein 33) and cysK-1 (encoding cysteine synthase A)

  • This genomic organization suggests functional connections between CoA metabolism, redox regulation, and cysteine biosynthesis

  • CoA serves as a major low-molecular-weight thiol in some bacteria, particularly those lacking glutathione

  • Type III PanKs are not subject to feedback inhibition by CoA, suggesting different regulatory mechanisms compared to Type I enzymes

  • Expression of bacterial Type III PanK genes is often upregulated during infection and early growth phases

  Understanding these metabolic interconnections is crucial for predicting potential compensatory mechanisms or combination drug targets that could enhance antimicrobial strategies targeting CoA biosynthesis.

Methodological Research Questions

• What are the optimal conditions for expressing and purifying recombinant Acinetobacter Type III PanK?

  Successful expression and purification of recombinant AbPanK requires optimization of multiple parameters:

  Expression System:

  • E. coli BL21(DE3) or similar expression strains are commonly used

  • Expression vectors containing T7 promoters (pET series) work effectively

  • Induction with IPTG (typically 0.5-1.0 mM) at OD₆₀₀ of 0.6-0.8

  • Post-induction growth at lower temperatures (18-25°C) often improves solubility

  Purification Protocol:

  • Affinity chromatography using His-tag (Ni-NTA) is effective for initial capture

  • Ion exchange chromatography for intermediate purification

  • Size exclusion chromatography for final polishing and buffer exchange

  • Typical buffers contain 50 mM Tris-HCl (pH 7.5-8.0), 100-300 mM NaCl, and 5-10% glycerol

  • Addition of 1-5 mM DTT or β-mercaptoethanol helps maintain enzyme activity

  Quality Control:

  • Dynamic light scattering to confirm homogeneity and determine hydrodynamic radius

  • SDS-PAGE should show a single band at approximately 30 kDa for AbPanK

  • Enzymatic activity assays to confirm functional protein using pantothenate and ATP as substrates

  Optimized expression and purification protocols typically yield 5-10 mg of pure protein per liter of bacterial culture, with >95% purity as assessed by SDS-PAGE.

• How can crystallization of Type III PanK be optimized for structural studies?

  Successful crystallization of Type III PanK requires careful optimization:

  Pre-crystallization Considerations:

  • Protein concentration typically between 10-15 mg/mL

  • High purity (>95%) confirmed by SDS-PAGE and size exclusion chromatography

  • Homogeneity confirmed by dynamic light scattering

  • Buffer optimization through thermal shift assays to identify stabilizing conditions

  Crystallization Approaches:

  • Sitting or hanging drop vapor diffusion methods

  • Initial screening using commercial sparse matrix screens

  • Optimization of hit conditions by varying:

    • Precipitant concentration

    • pH

    • Protein:reservoir ratio

    • Temperature (4°C vs. 20°C)

    • Additive screening

  Co-crystallization:

  • Addition of pantothenate (1-5 mM) often improves crystal quality

  • Co-crystallization with ATP/ADP can provide insights into binding modes

  • Non-hydrolyzable ATP analogs (AMP-PNP, ATPγS) useful for capturing pre-reaction state

  Post-crystallization:

  • Cryoprotection optimization (typically 20-25% glycerol, ethylene glycol, or PEG 400)

  • Selection of crystals showing sharp edges and uniform morphology

  • Diffraction quality assessment at home sources before synchrotron data collection

  AbPanK typically crystallizes in P2 space group with relatively large unit cell dimensions, requiring careful data collection strategies to resolve diffraction spots .

• What methods are most effective for assessing Type III PanK enzymatic activity?

  Several complementary approaches can be used to measure Type III PanK activity:

  Spectrophotometric Coupled Assays:

  • ADP production can be coupled to pyruvate kinase and lactate dehydrogenase reactions

  • NADH oxidation is monitored at 340 nm

  • Standard reaction conditions: 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 20 mM KCl, 1 mM ATP, 0.1-2 mM pantothenate, 0.3 mM NADH, 0.5 mM phosphoenolpyruvate, 5 units pyruvate kinase, 5 units lactate dehydrogenase

  Direct Phosphorylation Detection:

  • Radioactive assays using [γ-³²P]ATP to measure direct phosphoryl transfer

  • HPLC separation of pantothenate and 4′-phosphopantothenate

  • Malachite green assay for released inorganic phosphate

  Binding Studies:

  • Spectrofluorometric analyses to determine binding affinities (Kd) for pantothenate and ATP

  • Isothermal titration calorimetry for thermodynamic characterization

  • Surface plasmon resonance for real-time binding kinetics

  Kinetic Parameter Determination:

  • Determination of Km and kcat by varying substrate concentrations

  • Analysis of substrate inhibition or activation effects

  • pH and temperature dependence studies

  For AbPanK, spectrofluorometric binding studies have revealed a strong affinity for pantothenate (Kd = 1.2 × 10⁻⁸ M) and moderate affinity for ATP (Kd = 3.7 × 10⁻³ M) .

Comparative and Functional Questions

• How do Type III PanKs from different bacterial species compare in structure and function?

  Type III PanKs show both conservation and variation across bacterial species:

  Species	Molecular Weight	Key Structural Features	Regulatory Properties	Reference
  A. baumannii	~30 kDa	P2 space group, unique pantothenate binding pocket	No feedback inhibition by CoA
  B. anthracis	~30 kDa	Part of tricistronic operon with hslO and cysK-1	Essential for growth, no feedback inhibition
  P. aeruginosa	~30 kDa	Crystal structure available	No feedback inhibition
  T. maritima	~30 kDa	Thermostable variant, complexes with Pan and ADP	No feedback inhibition
  H. pylori	~30 kDa	One of the first characterized Type III PanKs	No feedback inhibition

  All Type III PanKs share the common feature of not being subject to feedback inhibition by CoA, unlike Type I enzymes . They also show a strong correlation with the absence of glutathione biosynthesis pathways in their respective organisms . Despite low sequence identity between different bacterial Type III PanKs (often <30%), their core catalytic domains maintain similar folding patterns and active site architectures .

  Crystal structures from various species have revealed common pantothenate and ATP binding modes, though with species-specific variations that could be exploited for selective inhibitor design .

• What is the genetic context of the coaX gene in Acinetobacter and how does it compare to other bacteria?

  The genetic context of the coaX gene provides insights into its regulation and functional associations:

  • In B. anthracis, coaX is the first gene in a tricistronic operon with hslO (encoding heat shock protein 33) and cysK-1 (encoding cysteine synthase A)

  • This coaX-hslO-cysK-1 cluster is conserved in several Bacillus species, Geobacillus kaustophilus, and some Listeria monocytogenes strains

  • In B. subtilis, a yacD gene (encoding a putative peptidyl-prolyl isomerase) is inserted within this cluster between hslO and cysK

  • Transcriptional profiling in B. anthracis shows that coaX, coaBC, and coaD genes (encoding the first three enzymes in the pantothenate→CoA pathway) are upregulated in early growth phases

  • The coaX gene is upregulated more than twofold between 1-2 hours post-infection within host macrophages

  This conserved genomic organization suggests functional connections between CoA metabolism, redox regulation (via Hsp33), and cysteine biosynthesis. The co-expression pattern during early growth and infection highlights the importance of CoA biosynthesis during bacterial proliferation and host adaptation, which further validates Type III PanK as an antimicrobial target.

• What genetic approaches have been used to validate Type III PanK as an essential enzyme?

  Multiple genetic approaches have confirmed the essentiality of Type III PanK:

  Conditional Mutants:

  • In B. anthracis, a temperature-dependent pNFd13 insertion mutagenesis method placed coaX under control of the IPTG-inducible Pspac promoter

  • Growth dependence on IPTG demonstrated essentiality, as cells failed to grow without the inducer

  • Suppressor mutations in the lac operator (e.g., G→A substitution at base pair 5) resulting in constitutive coaX expression further confirmed essentiality

  RNAi Approaches:

  • In Trypanosoma species, RNAi knockdown of PanK resulted in cell death which could be rescued by expression of heterologous PanK

  Gene Replacement:

  • Attempts at direct gene replacement without complementation typically fail, indicating essentiality

  • Successful gene replacement requires complementation with a functional copy of the gene

  Genomic Studies:

  • Genomic analyses consistently classify coaX as part of the minimal essential gene set in multiple bacterial species

  • Transposon insertion sequencing (Tn-seq) approaches typically show coaX as a gene that cannot tolerate insertions

  These genetic validations, particularly from conditional mutant studies, provide strong evidence that Type III PanK is essential for bacterial viability and confirm its potential as an antimicrobial target .

• How can potential inhibitors of Type III PanK be evaluated in experimental settings?

  A comprehensive evaluation of Type III PanK inhibitors involves multiple experimental approaches:

  Biochemical Assays:

  • Enzyme inhibition assays using purified recombinant protein to determine IC₅₀ and Ki values

  • Mechanism of inhibition studies (competitive, non-competitive, uncompetitive, or mixed)

  • Binding affinity measurements using spectrofluorometry, ITC, or SPR

  • Assessment of selectivity against different PanK types (I, II, and III) from various species

  Structural Studies:

  • Co-crystallization of inhibitors with PanK to determine binding modes

  • Structure-activity relationship analysis to guide optimization

  • Molecular dynamics simulations to understand binding dynamics

  Cellular Assays:

  • Determination of minimum inhibitory concentrations (MICs) against Acinetobacter and other bacteria with Type III PanKs

  • Metabolomic profiling to confirm on-target effects (reduced CoA levels)

  • Cytotoxicity testing against mammalian cells to assess selectivity

  • Time-kill kinetics to characterize bactericidal vs. bacteriostatic activity

  Resistance Studies:

  • Selection of resistant mutants and whole-genome sequencing to identify resistance mechanisms

  • Construction of target overexpression strains to confirm on-target activity

  • Assessment of resistance development frequency

  In Vivo Evaluation:

  • Pharmacokinetic studies to assess ADME properties

  • Efficacy in animal infection models

  • Toxicity and safety evaluations

  The most promising inhibitors would demonstrate potent activity against purified enzyme (nanomolar IC₅₀), selective inhibition of bacterial over human PanKs, good correlation between enzyme inhibition and antibacterial activity, and efficacy in animal infection models with acceptable safety profiles.