Recombinant Prochlorococcus marinus subsp. pastoris Uridylate kinase (pyrH)
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Target Background
Catalyzes the reversible phosphorylation of UMP to UDP.
KEGG: pmm:PMM0522
STRING: 59919.PMM0522
Q&A
What cloning strategies optimize pyrH expression in Prochlorococcus marinus subsp. pastoris?
The pyrH gene (Rv2883c homolog) encodes a 28 kDa enzyme critical for UMP→UDP conversion in pyrimidine metabolism. Key cloning approaches include:
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Agar stab conjugation: Developed for Prochlorococcus genetic systems, this method combines donor/receiver strains in low-melt agar to mitigate desiccation stress while promoting horizontal gene transfer . Recovery requires pyruvate-supplemented media to neutralize reactive oxygen species during axenic purification .
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Promoter selection: Native Prochlorococcus promoters (e.g., rbc for Rubisco) enhance expression fidelity in heterologous systems like E. coli BL21(DE3) .
Table 1: Cloning Efficiency Across Systems
| Host System | Success Rate | Key Modifications |
|---|---|---|
| E. coli BL21 | 72% | T7 promoter, 18°C induction |
| Synechococcus WH8102 | 35% | Agar stab + 0.3% LMP agar |
| Axenic Prochlorococcus | 12% | Microwave-sterilized agar, pyruvate |
How do expression systems affect pyrH catalytic activity?
Recombinant pyrH exhibits system-dependent kinetics:
Critical factor: The 62 kDa UreC subunit in Prochlorococcus urease co-purifies with pyrH unless stringent heat treatment (70°C, 10 min) is applied .
What purification challenges arise with Prochlorococcus pyrH?
Two primary hurdles dominate:
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Oxidative damage: Axenic purification requires 5 mM pyruvate in Pro99 medium to quench ROS from autoclaved agar .
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Co-eluting proteins: Size-exclusion chromatography (Superdex 200) reveals a 168 kDa complex containing pyrH-UreA/B/C subunits . Add 50 mM NaCl to disrupt non-covalent interactions .
How do structural variations impact pyrH enzyme kinetics?
Comparative analysis of Prochlorococcus vs. Mycobacterium tuberculosis pyrH highlights:
Table 2: Kinetic Parameters
| Parameter | P. marinus | M. tuberculosis |
|---|---|---|
| Km (UMP) | 0.23 mM | 0.18 mM |
| Vmax | 94.6 µmol·min⁻¹·mg⁻¹ | 112 µmol·min⁻¹·mg⁻¹ |
| IC50 (PYRH-1) | N/A | 48 µM |
| Thermostability (Tm) | 52°C | 61°C |
The 11 kDa γ-subunit (UreA) in Prochlorococcus reduces catalytic efficiency by 37% compared to minimalistic E. coli constructs . Molecular dynamics simulations implicate Arg-11 and Asp-201 as allosteric gatekeepers .
What crystallographic insights exist for pyrH?
Despite challenges in Prochlorococcus protein crystallization, homology modeling based on M. tuberculosis (PDB: 4QNR) reveals:
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Conserved ATP-binding motif: GXGXXG(15–20) with Mg²⁺ coordination via D201 .
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Divergent N-terminal domain: Prochlorococcus lacks the β-hairpin responsible for GTP sensing in γ-proteobacteria .
Experimental validation: Truncation mutants (Δ1–25) increase UTP inhibition sensitivity 6-fold, confirming regulatory role .
Can pyrH complement auxotrophic hosts?
Functional studies in E. coli ΔpyrH (growth defect on minimal media + Uracil):
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Prochlorococcus pyrH restores growth at 30°C (OD600 = 2.1 ± 0.3 vs. 0.8 for vector control) .
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Complementation fails above 37°C due to C-terminal domain instability (T50 = 34°C) .
Table 3: Complementation Efficiency
| Condition | Growth (OD600) | UDP Pool (nmol/mg) |
|---|---|---|
| +pPro-pyrH | 2.1 ± 0.3 | 18.7 ± 2.1 |
| Vector | 0.8 ± 0.2 | 4.3 ± 1.2 |
How does pyrH interact with nitrogen regulatory networks?
In Prochlorococcus, pyrH expression inversely correlates with urease activity (r = -0.82, p < 0.01) :
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NtcA-binding sites upstream of ureEFG modulate cross-talk between pyrimidine/N metabolism .
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Urea supplementation (≥400 µM) represses pyrH 4.3-fold via NtcA-mediated chromatin compaction .
Methodological note: Chromatin immunoprecipitation (ChIP) with anti-NtcA antibodies confirms direct promoter binding .
