Engineering of Phycourobilin Synthase: PubS to a Two-Electron Reductase

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Phycourobilin:ferredoxin oxidoreductase (PubS) is a ferredoxin-dependent bilin reductase (FDBR) enzyme.
• PubS catalyzes the reduction of biliverdin IXα to produce phycourobilin, specifically reducing the C15=C16 and C4=C5 double bonds.
• Unlike other FDBRs, PubS is unique in reducing the C4=C5 double bond and belongs to the four-electron reduction subclass.

Purpose of the Study:

• To investigate the role of arginine residues in substrate binding and reduction mechanisms of FDBR enzymes.
• To understand the molecular basis for the distinct substrate binding modes observed in FDBRs.
• To engineer PubS to alter its reduction mechanism and gain insights into chromophore binding and proton donation.

Main Methods:

• Sequence alignment and comparison of PubS with other FDBRs to identify key residues.
• Site-directed mutagenesis of PubS to alter substrate binding and reduction properties.
• Biochemical characterization of wild-type and mutant PubS enzymes to assess their catalytic activity and product profiles.

Main Results:

• Identified a key arginine residue in PubS analogous to those in two-electron reduction FDBRs.
• Mutagenesis successfully converted PubS from a four-electron to a two-electron reduction enzyme.
• Observed accumulation of radicals in the modified PubS, indicating a shift in the reduction mechanism.

Conclusions:

• The study provides a molecular understanding of chromophore binding modes and proton donation in FDBR enzymes.
• Modification of PubS offers a novel approach to study enzyme mechanisms and engineer enzyme function.
• The findings contribute to the broader knowledge of bilin metabolism and FDBR enzyme evolution.