Engineering a trifunctional proline utilization A chimaera by fusing a DNA-binding domain to a bifunctional PutA

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Proline utilization A (PutA) is a crucial flavoenzyme in proline metabolism.
• Trifunctional PutA enzymes possess DNA-binding domains and regulate gene expression.
• These enzymes exhibit unique functional switching between DNA binding and enzymatic activity.

Purpose of the Study:

• To construct a trifunctional PutA by fusing the RHH domain of E. coli PutA to R. capsulatus PutA.
• To investigate the modular design and functional switching capabilities of PutA.
• To explore the role of the C-terminal domain in PutA's functional switching.

Main Methods:

• Gene fusion of the Escherichia coli PutA RHH domain with Rhodobacter capsulatus PutA.
• Biochemical assays to assess catalytic activity and DNA-binding properties.
• Lipid association studies to evaluate membrane association.

Main Results:

• The EcRHH-RcPutA chimera retained catalytic activity and acquired DNA-binding properties.
• The chimera demonstrated proline-induced lipid association, a hallmark of functional switching.
• Bifunctional PutA also showed proline-activated lipid binding, indicating limited functional switching.

Conclusions:

• The study successfully constructed a hybrid PutA enzyme with dual functions.
• Functional switching in PutA is modular and influenced by proline availability.
• The C-terminal domain is critical for the functional switching mechanism in trifunctional PutA.