Discovery of posttranslational maturation by self-subunit swapping

Area of Science:

• Biochemistry
• Enzymology
• Microbial Metabolism

Background:

• General metallocenter biosynthesis involves apoprotein subunits remaining in the final holoprotein.
• The cobalt-containing low-molecular-mass nitrile hydratase (L-NHase) from Rhodococcus rhodochrous J1 is composed of beta- and alpha-subunits (nhlBA genes).
• An open reading frame, nhlE, downstream of nhlBA, is essential for L-NHase activation.

Purpose of the Study:

• To elucidate the novel mechanism of L-NHase activation and cobalt incorporation.
• To investigate the role of nhlE in the maturation of cobalt-containing L-NHase.
• To characterize the difference between L-NHase derived from nhlBA and nhlBAE.

Main Methods:

• Genetic analysis of nhlBA and nhlE genes.
• Biochemical characterization of apo-L-NHase and holo-L-NHase.
• Identification and characterization of the L-NHase maturation mediator, NhlAE.

Main Results:

• Gene products from nhlBA yield cobalt-free apo-L-NHase lacking oxidized cysteine residues.
• Gene products from nhlBAE yield cobalt-containing holo-L-NHase with Cys-SO(2)(-) and Cys-SO(-) metal ligands.
• NhlAE, comprising NhlE and the modified alpha-subunit, acts as a maturation mediator.
• Cobalt incorporation depends on the exchange of the apo-L-NHase alpha-subunit with the NhlAE alpha-subunit.

Conclusions:

• A novel posttranslational maturation process, termed "self-subunit swapping," was discovered.
• This process involves the exchange of an apo-L-NHase subunit with a modified subunit from a mediator complex.
• This mechanism differs significantly from established metallocenter biosynthesis pathways.