Reconstitution of Formylglycine-generating Enzyme with Copper(II) for Aldehyde Tag Conversion.
To further our aim of synthesizing aldehyde-tagged proteins for research and biotechnology applications, we developed methods for recombinant production of aerobic formylglycine-generating enzyme (FGE) in good yield. We then optimized the FGE biocatalytic reaction conditions for conversion of cysteine to formylglycine in aldehyde tags on intact monoclonal antibodies. During the development of these conditions, we discovered that pretreating FGE with copper(II) is required for high turnover rates and yields. After further investigation, we confirmed that both aerobic prokaryotic (Streptomyces coelicolor) and eukaryotic (Homo sapiens) FGEs contain a copper cofactor. The complete kinetic parameters for both forms of FGE are described, along with a proposed mechanism for FGE catalysis that accounts for the copper-dependent activity.
From Catalent Pharma Solutions, Emeryville, California 94608.
Patrick G
PG
Holder
From Catalent Pharma Solutions, Emeryville, California 94608.
Lesley C
LC
Jones
From Catalent Pharma Solutions, Emeryville, California 94608.
Penelope M
PM
Drake
From Catalent Pharma Solutions, Emeryville, California 94608.
Robyn M
RM
Barfield
From Catalent Pharma Solutions, Emeryville, California 94608.
Stefanie
S
Bañas
From Catalent Pharma Solutions, Emeryville, California 94608.
Gregory W
GW
de Hart
From Catalent Pharma Solutions, Emeryville, California 94608.
Jeanne
J
Baker
From Catalent Pharma Solutions, Emeryville, California 94608. Electronic address: david.rabuka@catalent.com.
David
D
Rabuka
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003067:Coenzymes / Q000737:chemistry* / Q000378:metabolism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D050862:Oxidoreductases Acting on Sulfur Group Donors; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics; D013429:Sulfatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D003067:Coenzymes; D003300:Copper; D050862:Oxidoreductases Acting on Sulfur Group Donors; C480483:SUMF1 protein, human; D013429:Sulfatases; D003545:Cysteine
aldehyde tag; antibody drug conjugation; catalysis; enzyme kinetics; metalloenzyme; monoclonal antibody; post-translational modification (PTM)