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Towards a functional identification of catalytically inactive [Fe]‐hydrogenase paralogs
Author(s) -
Fujishiro Takashi,
Ataka Kenichi,
Ermler Ulrich,
Shima Seigo
Publication year - 2015
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13351
Subject(s) - cofactor , hydrogenase , archaea , enzyme , chemistry , biochemistry , tyrosine , histidine , stereochemistry , gene
[Fe]‐hydrogenase (Hmd), an enzyme of the methanogenic energy metabolism, harbors an iron‐guanylylpyridinol (Fe GP ) cofactor used for H 2 cleavage. The generated hydride is transferred to methenyl‐tetrahydromethanopterin (methenyl‐H 4 MPT + ). Most hydrogenotrophic methanogens contain the hmd ‐related genes hmd II and hmd III . Their function is still elusive. We were able to reconstitute the Hmd II holoenzyme of Methanocaldococcus jannaschii with recombinantly produced apoenzyme and the Fe GP cofactor, which is a prerequisite for in vitro functional analysis. Infrared spectroscopic and X‐ray structural data clearly indicated binding of the Fe GP cofactor. Methylene‐H 4 MPT binding was detectable in the significantly altered infrared spectra of the Hmd II holoenzyme and in the Hmd II apoenzyme–methylene‐H 4 MPT complex structure. The related binding mode of the Fe GP cofactor and methenyl‐H 4 MPT + compared with Hmd and their multiple contacts to the polypeptide highly suggest a biological role in Hmd II . However, holo‐Hmd II did not catalyze the Hmd reaction, not even in a single turnover process, as demonstrated by kinetic measurements. The found inactivity can be rationalized by an increased contact area between the C‐ and N‐terminal folding units in Hmd II compared with in Hmd, which impairs the catalytically necessary open‐to‐close transition, and by an exchange of a crucial histidine to a tyrosine. Mainly based on the presented data, a function of Hmd II as Hmd isoenzyme, H 2 sensor, Fe GP ‐cofactor storage protein and scaffold protein for Fe GP ‐cofactor biosynthesis could be excluded. Inspired by the recently found binding of Hmd II to aminoacyl‐ tRNA synthetases and tRNA , we tentatively consider Hmd II as a regulatory protein for protein synthesis that senses the intracellular methylene‐H 4 MPT concentration. Database Structural data are available in the Protein Data Bank under the accession numbers 4YT8 ; 4YT2 ; 4YT4 and 4YT5 .

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