Solution Structure and Backbone Dynamics of an Endopeptidase HycI from scherichia coli

Abstract

[NiFe] hydrogenases are metalloenzymes involved in many biological processes concerning the metabolism of hydrogen. The maturation of the large subunit of these hydrogenases requires the cleavage of a peptide at the C terminus by an endopeptidase before the final formation of the [NiFe] metallocenter. HycI is an endopeptidase of the M52 family and responsible for the C-terminal cleavage of the large subunit of hydrogenase 3 in Escherichia coli. Although extensive studies were performed, the molecular mechanism of recognition and cleavage of hydrogenase 3 remains elusive. Herein, we report the solution structure of E. coli HycI determined by high resolution nuclear magnetic resonance spectroscopy. This is the first solution structure of the apo form of endopeptidase of the M52 family reported thus far. The overall structure is similar to the crystal structure of holo-HybD in the same family. However, significant diversity was observed between the two structures. Especially, HycI shows an open conformation at the putative nickel-binding site, whereas HybD adopts a closed conformation. In addition, we performed backbone dynamic studies to probe the motional properties of the apo form of HycI. Furthermore, the metal ion titration experiments provide insightful information on the substrate recognition and cleavage processes. Taken together, our current structural, biochemical, and dynamic studies extend the knowledge of the M52 family proteins and provide novel insights into the biological function of HycI.Hydrogenases are generally metal-containing enzymes and play a central role in the hydrogen metabolism of many microorganisms. They are involved in many biological processes in which hydrogen molecules are produced and consumed. Based on their metal contents at the active site, metallohydrogenases are classified into two main types, the [Fe] and [NiFe] hydrogenases (1). Escherichia coli has four different [NiFe] hydrogenases, designated as hydrogenases 1-4. Hydrogenases 1-3 are responsible for anaerobic H 2 oxidation for energy conserving or involved in H 2 production from formate, whereas the function of hydrogenase 4 remains elusive (2-4). Generally, they are heterodimers consisting of small and large subunits. When initially synthesized in cells, the large subunit is in the precursor form lacking the metallocenter and the [NiFe] center is subsequently formed by a complex maturation process. For E. coli hydrogenase 3, seven proteins encoded by genes hypABCDE, hypF, and hycI are involved in the regulation and maturation of the large subunit (HycE) (5, 6). Among them, three proteins responsible for the nickel incorporation play key roles in this process. The HypB protein exhibits GTPase activity and is responsible for nickel delivery (7), the HypC protein acts as a chaperone that interacts with the pre-HycE to form an open conformation for the metal insertion (8), and finally HycI, an endopeptidase, cleaves the C terminus of the pre-HycE. Subsequently, the formation of the metallocenter associated with a conforma...

Identification of a stable complex between a [NiFe]‐hydrogenase catalytic subunit and its maturation protease

“…The crystal structures of processing proteases revealed metal‐binding enzymes. E. coli HybD, for example, contains cadmium ions in the structure , and E. coli HycI has a calcium ion binding site . Proteolytic cleavage of the large subunit by the endopeptidase has been considered ‘nickel dependent’ in so far as the [NiFe] cofactor must be loaded into the large subunit before proteolysis .…”

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostocsp. strain PCC 7120

“…This novel observation of a conserved group specific region may be an important finding for the understanding of the specificity and function of hydrogenase specific proteases. The function of this region in hydrogenase specific proteases has previously been under speculation with some suggesting that it functions as a catalytic site for the proteolytic cleavage [17,61] and others that it is involved in substrate binding [17]. Amino acid replacement, whereby Asp38 in HycI in E. coli was changed to an asparagine showed no effect on the cleavage process [62] which of course does not rule out that other parts of this region might be of importance.…”

Metabolically engineered bacteria for producing hydrogen via fermentation

“…The crystal structures of HypB from M. jannaschii (Gasper et al ., 2006), HypC, HypD and HypE from Thermococcus kodakaraensis (Watanabe et al ., 2007), HybD from E. coli (Fritsche et al ., 1999), HypF N‐terminal acylphosphatase domain (residues 1–91) from E. coli (Rosano et al ., 2002), and the NMR solution structure of HycI from E. coli (Yang et al ., 2007) are known (Table 3) and provide insight into their functions in maturation process.…”