Structure of [NiFe] hydrogenase maturation protein HypE from Escherichia coli and its interaction with HypF

DOIResolve DOI: http://doi.org/10.1128/JB.01610-07
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TypeArticle
Volume190
Issue4
Pages14471458; # of pages: 12
SubjectChromatography; crystal structure; Enzymes; Escherichia coli; Ligands; metabolism; Molecular Weight; Operon; pha; Protein; Proteins; Surface Plasmon Resonance; synthesis
AbstractHydrogenases are enzymes involved in hydrogen metabolism, utilizing H2 as an electron source. [NiFe] hydrogenases are heterodimeric Fe-S proteins, with a large subunit containing the reaction center involving Fe and Ni metal ions and a small subunit containing one or more Fe-S clusters. Maturation of the [NiFe] hydrogenase involves assembly of nonproteinaceous ligands on the large subunit by accessory proteins encoded by the hyp operon. HypE is an essential accessory protein and participates in the synthesis of two cyano groups found in the large subunit. We report the crystal structure of Escherichia coli HypE at 2.0-A resolution. HypE exhibits a fold similar to that of PurM and ThiL and forms dimers. The C-terminal catalytically essential Cys336 is internalized at the dimer interface between the N- and C-terminal domains. A mechanism for dehydration of the thiocarbamate to the thiocyanate is proposed, involving Asp83 and Glu272. The interactions of HypE and HypF were characterized in detail by surface plasmon resonance and isothermal titration calorimetry, revealing a Kd (dissociation constant) of approximately 400 nM. The stoichiometry and molecular weights of the complex were verified by size exclusion chromatography and gel scanning densitometry. These experiments reveal that HypE and HypF associate to form a stoichiometric, hetero-oligomeric complex predominantly consisting of a [EF]2 heterotetramer which exists in a dynamic equilibrium with the EF heterodimer. The surface plasmon resonance results indicate that a conformational change occurs upon heterodimerization which facilitates formation of a productive complex as part of the carbamate transfer reaction
Publication date
AffiliationNRC Biotechnology Research Institute; National Research Council Canada
Peer reviewedNo
NRC number49530
NPARC number3539693
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Record identifier22cfddc2-0029-4ac0-8cba-e4baef7c301c
Record created2009-03-01
Record modified2016-05-09
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