Recombinant Pseudomonas aeruginosa Protein nirD (nirD)
Description
Introduction to Recombinant Pseudomonas aeruginosa Protein nirD (nirD)
The nirD gene in Pseudomonas aeruginosa encodes a protein critical for the biosynthesis of heme d₁, a unique tetrapyrrole cofactor essential for the dissimilatory nitrite reductase (NirS) enzyme. NirD functions as part of the nirFDLGHJE gene cluster, which collectively regulates heme d₁ production and maturation . This article synthesizes current research on the role of recombinant nirD in bacterial denitrification, its biochemical properties, and its integration into the broader denitrification pathway.
Heme d₁ Biosynthesis
NirD operates in the final stages of heme d₁ biosynthesis, a process initiated by the uroporphyrinogen III methyltransferase NirE (PA0510) . The pathway involves sequential modifications to yield the unique acrylate side chains of heme d₁, which are critical for NirS activity . While specific enzymatic functions of NirD remain under investigation, homology studies suggest it may act as a decarboxylase, removing carboxyl groups during tetrapyrrole maturation .
Denitrification Pathway Integration
NirD’s role is upstream of NirS, the periplasmic nitrite reductase that converts nitrite (NO₂⁻) to nitric oxide (NO) in the denitrification cascade . Defects in heme d₁ biosynthesis, including those caused by nirD disruption, impair NirS activity, leading to incomplete denitrification and accumulation of reactive nitrogen intermediates .
Recombinant Production and Purification
Recombinant nirD is typically expressed in heterologous systems such as E. coli BL21(DE3) using expression vectors like pET-22b(+) . Purification involves affinity chromatography (e.g., His-tagged systems) and SDS-PAGE validation. Structural studies of recombinant NirD remain limited, but its homology to other cluster proteins (e.g., NirL, NirG) suggests a globular fold stabilized by conserved motifs .
Table 1: NirD Functional Context in the nirFDLGHJE Cluster
Table 2: Heme d₁ Biosynthesis Pathway
| Step | Substrate | Product | Enzyme(s) Involved |
|---|---|---|---|
| 1 | Uroporphyrinogen III | Siroheme | NirE, NirDL |
| 2 | Siroheme | Dihydro-heme d₁ | NirG, NirH |
| 3 | Dihydro-heme d₁ | Heme d₁ | NirN |
Implications for Denitrification
NirD’s disruption leads to defective heme d₁ biosynthesis, impairing NirS activity. This results in reduced nitrite-to-NO conversion efficiency, as demonstrated in P. aeruginosa ΔnirN mutants . Such defects compromise the bacterium’s ability to thrive in anaerobic environments, emphasizing nirD’s critical role in maintaining energy homeostasis .
Product Specs
Target Background
KEGG: pae:PA0515
STRING: 208964.PA0515
