Receivedforpublication,March19,2015,andinrevisedform,April27,2015 Published,JBCPapersinPress,April30,2015,DOI10.1074/jbc.M115.652669
Patrick G. Holder, Lesley C. Jones, Penelope M. Drake, Robyn M. Barfield, Stefanie Ban ̃ as, Gregory W. de Hart, Jeanne Baker, and David Rabuka1
From Catalent Pharma Solutions, Emeryville, California 94608
http://www.jbc.org/content/290/25/15730.full?sid=c7d620e4-30fa-46a4-8b1c-4fbd922f5d95
Background: Aerobic formylglycine-generating enzyme (FGE) converts cysteine to formylglycine in vivo.
Results: Purified FGE requires preactivation with copper to convert cysteine to formylglycine in vitro.
Conclusion: FGE is a metalloenzyme. It is also a useful biocatalyst for the production of proteins that contain aldehyde tags. Significance: Understanding FGE biochemistry informs research on sulfatases and enables expanded biotechnology applica- tions of the aldehyde tag.
Patrick G. Holder, Lesley C. Jones, Penelope M. Drake, Robyn M. Barfield, Stefanie Ban ̃ as, Gregory W. de Hart, Jeanne Baker, and David Rabuka1
From Catalent Pharma Solutions, Emeryville, California 94608
http://www.jbc.org/content/290/25/15730.full?sid=c7d620e4-30fa-46a4-8b1c-4fbd922f5d95
Background: Aerobic formylglycine-generating enzyme (FGE) converts cysteine to formylglycine in vivo.
Results: Purified FGE requires preactivation with copper to convert cysteine to formylglycine in vitro.
Conclusion: FGE is a metalloenzyme. It is also a useful biocatalyst for the production of proteins that contain aldehyde tags. Significance: Understanding FGE biochemistry informs research on sulfatases and enables expanded biotechnology applica- tions of the aldehyde tag.
Abstract:
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 antibod-
ies. During the development of these conditions, we discovered
that pretreating FGE with copper(II) is required for high turn-
over 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.
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