Recombinant Geobacillus stearothermophilus Glutamine amidotransferase subunit pdxT (pdxT)

Introduction

The recombinant Geobacillus stearothermophilus glutamine amidotransferase subunit pdxT is a critical component of the pyridoxal 5'-phosphate synthase (PLPS) complex, essential for de novo vitamin B6 biosynthesis in thermophilic organisms. This enzyme catalyzes the hydrolysis of glutamine to produce ammonia, which is subsequently utilized by the synthase subunit pdxS to synthesize pyridoxal 5'-phosphate (PLP), a cofactor vital for numerous metabolic pathways. The recombinant expression of G. stearothermophilus pdxT in heterologous systems like Escherichia coli has enabled detailed biochemical and structural characterization, highlighting its potential for industrial applications .

Catalytic Mechanism

PdxT belongs to the class I glutamine amidotransferase family and employs a conserved Cys-His-Glu catalytic triad to hydrolyze glutamine. This reaction releases ammonia, which is transferred to pdxS for PLP synthesis. The spatial separation of catalytic functions between pdxT (ammonia production) and pdxS (PLP formation) ensures efficient coupling of these steps .

Subunit Interaction

In the PLPS complex, pdxT interacts with pdxS in a 12:12 subunit ratio, forming a 24-subunit structure. PdxT subunits dock on the outer surface of pdxS dodecamers, aligning their active sites to facilitate ammonia transfer. This structural organization is critical for enzymatic activity and is conserved across thermophilic Geobacillus species .

Cloning and Expression

The pdxT gene from Geobacillus sp. H6a (a thermophilic strain closely related to G. stearothermophilus) was cloned into the pET28a vector, introducing a His-tag for affinity purification. Expression in E. coli BL21(DE3) required optimization:

• IPTG concentration: 0.5 mM induced maximal soluble protein expression.

• Temperature: 30°C improved solubility compared to 37°C .

Purification and Activity Assay

Purified pdxT was assayed using a two-step method:

1. Glutamine hydrolysis: Ammonia release was quantified via glutamic dehydrogenase-coupled reactions .

2. PLP synthesis: Activity was confirmed by combining pdxT with pdxS and monitoring PLP production .

Kinetic Parameters

The recombinant pdxT exhibited the following kinetic properties when paired with pdxS:

Metal Ion Sensitivity

Divalent cations modulate pdxT activity:

• Enhancers: Mg²⁺ (50% increase), Ca²⁺ (30% increase) .

• Inhibitors: Fe³⁺, Cu²⁺ (complete inhibition at 1 mM) .

Stability

PdxT retained ~80% activity after exposure to:

• High temperatures: 70°C for 1 hour.

• Broad pH ranges: 6.0–10.0 .

Quaternary Structure

PdxT exists as a monomer in solution, contrasting with pdxS, which forms hexamers/dodecamers. The PLPS complex assembles into a 24-subunit structure (12 pdxS + 12 pdxT), with pdxT subunits positioned externally to facilitate ammonia transfer .

Catalytic Triad Conservation

The Cys-His-Glu triad in pdxT is conserved across Geobacillus species, including G. stearothermophilus, ensuring functional fidelity despite thermal stress .

Applications in Industrial Biotechnology

The thermostability of G. stearothermophilus pdxT makes it ideal for high-temperature industrial processes:

• Vitamin B6 production: PLPS-driven biosynthesis could replace chemical synthesis methods.

• Biocatalytic systems: PdxT’s ammonia production could be integrated into multi-enzyme cascades .