Nucleotide sequence and expression of an operon in Escherichia coli coding for formate hydrogenylase components

Abstract

An 8kb segment of DNA from the 58/59 min region of the E. coli chromosome, which complements the defect of a mutant devoid of hydrogenase 3 activity, has been sequenced. Eight open reading frames were identified which are arranged in a transcriptional unit; all open reading frames were transcribed and translated in vivo in a T7 promoter/polymerase system. Analysis of the amino acid sequences derived from the nucleic acid sequences revealed that one of them, open reading frame 5 (ORF5), exhibits significant sequence similarity to conserved regions of the large subunit from Ni/Fe hydrogenases. Two of the open reading frames (orf2, orf6) code for proteins apparently carrying iron-sulphur clusters of the 4Fe/4S ferredoxin type. The product of one of the open reading frames, orf7, displays extensive sequence similarity with protein G from the chloroplast electron transport chain. ORF3 and ORF4, on the other hand, are extremely hydrophobic proteins with nine and six putative transmembrane helices, respectively. Over a limited hydrophilic sequence stretch, bordered by putative transmembrane areas, ORF3 and ORF4 exhibit homology with subunits 4 and 1 of mitochondrial and plastid NADH-ubiquinol oxidoreductases, respectively. The operon described, therefore, appears to comprise genes for redox carriers linking formate oxidation to proton reduction and for a hydrogenase of hitherto unique composition.

Reassignment of the gene encoding the Escherichia coli hydrogenase 2 small subunit

“…Cultures were harvested by centrifugation at 7000 g, 4°C, for 15 min, the cell pellet was washed twice with ice-cold 100 mM potassium phosphate, pH 7.0, and resedimented as described above. Washed cells were either stored as pellets at Ϫ20°C, or resuspended in 50 mM Tris/ HCl, pH 7.5, 100 mM KCl, 1 mM benzamidine/HCL, 1 mM MgSO 4 . A crystal of deoxyribonuclease I (Sigma) was added to this, and the cells were broken by passage through a French pressure cell (2100 Pa).…”

“…Hydrogenase 3 is encoded by an operon (hyc) located at 58 min on the E. coli genome, which is composed of nine open reading frames hycABCDEF-GHI [4,10]. The HycE gene encodes the nickel-containing large subunit.…”

Properties of the [NiFe]‐hydrogenase maturation protein HypD

“…The synthesis and insertion of the NiFe(CO)(CN) 2 metal centre depends on the activities of a series of proteins. The core machinery, as demonstrated for the synthesis of the intracellularly located hydrogenase 3 from E. coli, consists of the products of the six hyp genes plus an endopeptidase ( [1][2][3][4] and for review see [5,6]). The two hyp gene products HypA and HypB together with the auxiliary protein SlyD [7][8][9] are required for nickel sequestration and incorporation, whereas the four other Hyp proteins (HypC to HypF) are involved in synthesis of the CN ligand and the cyanation of the active site iron [10][11][12][13].…”

Infrared-Detectable Group Senses Changes in Charge Density on the Nickel Center in Hydrogenase from Chromatium vinosum

“…Halboth & Klein, 1992), usually associated with two regular cubane clusters. In two enzymes, the six Cys residues are missing altogether, and the amino acid sequence of the small subunit terminates shortly after the first four conserved residues (Bohm et al, 1990;Tran-Betcke et al, 1990). This indicates that the clusters coordinated by these Cys residues are not essential for hydrogen activation.…”