coli in which all three Hyd maturation proteins are co-expressed and has been interpreted to indicate that a protein associated intermediate in cluster biosynthesis is generated under these conditions and readily transferred to the hydA gene product to accomplish activation.įollowing this development, we have pursued the identification of the activating component present in the cell extract. The in vitro activation of HydA occurs only with extracts of E. acetobutylicum are expressed simultaneously can activate heterologously expressed HydA. coli in which all three Hyd proteins from C. In our most recent work, we have shown that cell extracts of E. Preliminary biochemical characterization of heterologously expressed HydE, HydF, and HydG from Thermatoga maritima corroborated the general functional inferences derived from genomic analysis and showed that all three proteins are capable of binding FeS clusters. Genome annotation indicates that HydE and HydG are members of the radical S-adenosylmethionine (AdoMet) enzyme family, and that HydF is likely to be a GTPase. Further studies have shown that the coexpression in Escherichia coli of HydE, HydF, and HydG from Clostridium acetobutylicum with the HydA hydrogenase protein from a variety of microbial sources enables the formation of active hydrogenases. Recent studies involving the analysis of mutants of Chlamydomonas reinhardtii defective in hydrogen production have revealed that products of the hydEF and hydG genes are required for the accumulation of active hydrogenase, and that hydE, hydF, and hydG are common to all organisms in which active hydrogenases are found. While progress has been made in defining the role of specific gene products and identifying the precursors of the non-protein ligands in the hydrogenases relatively little is known about maturation of the “H-cluster” of the hydrogenases. In addition to these ligands, the irons of the subcluster are bridged by a non-protein dithiolate linkage. The hydrogenase active site consists of the “H-cluster” which exists as a cluster linked by a cysteinyl thiolate to a di-iron subcluster coordinated by carbon monoxide and cyanide ligands. The hydrogenase active site consists of an iron atom ligated by one carbon monoxide and two cyanide ligands bridged to a nickel atom via two cysteine thiolates.
These strong field ligands presumably function to stabilize low oxidation states of iron. Although phylogenetically unrelated, these enzymes share biologically unique sites containing iron with both cyanide and carbon monoxide ligands. © 2010 The Authors Journal compilation © 2010 FEBS.The and hydrogenases catalyze hydrogen oxidation and proton reduction and function either to couple hydrogen oxidation to energy yielding processes or to regenerate reduced electron carriers accumulated during fermentation. Thus, the field is in a state of continuous advance and expansion, which demands that the classification scheme be regularly updated and, if needed, revised. In addition, although not usually considered as scaffolds, several other proteins, such as regulatory proteins with catalytic activity, phosphatase targeting subunits, E3 ubiquitin ligases, ESCRT proteins in endosomal sorting and DNA damage sensors also function by bona fide scaffolding mechanisms. It will also be shown, however, that the categories partially overlap and often their names are used interchangeably in the literature.
Here we discuss the categories of scaffolds, anchors, docking proteins and adaptors in some detail, and using many examples we demonstrate that they cover a wide range of functional modes that appear to segregate into three practical categories, simple proteins binding two partners together (adaptors), larger multidomain proteins targeting and regulating more proteins in complex ways (scaffold/anchoring proteins) and proteins specialized to initiate signaling cascades by localizing partners at the cell membrane (docking proteins). By binding and bringing into proximity two or more signaling proteins, these proteins direct the flow of information in the cell by activating, coordinating and regulating signaling events in regulatory networks. In this series of four minireviews the field of scaffold proteins and proteins of similar molecular/cellular functions is overviewed.