Crystallization and preliminary X-ray characterization of the 2,4'-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP.

The enzyme 2,4'-dihydroxyacetophenone dioxygenase (or DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.

Presence and role of a second disulphide bond in recombinant lupanine hydroxylase using site-directed mutagenesis with 143Cys→Ser and 124,143Cys→Ser mutations in Escherichia coli.

Lupanine hydroxylase (LH), a quinohaemoprotein, catabolizes lupanine and possesses four cysteine (Cys) residues; two associated with a cytochrome c motif ((586)Cys and (589)Cys), while the role of the remaining two residues ((124)Cys and (143)Cys) is unclear. Structural graphic simulation using homology modelling suggested a potential second -S-S- bond, a common feature between adjacent Cys residues in other quinohaemoproteins; however, in LH, these residues are located 18 amino acids apart. Formation of the second disulphide bond was initially chemically confirmed by iodomethane alkylation with 91% loss of enzymic activity, and no significant change was observed with unreduced alkylated protein.

Export of a hyperexpressed mammalian globular cytochrome b5 precursor in Escherichia coli is dramatically affected by the nature of the amino acid flanking the secretory signal sequence cleavage bond.

A chimeric mammalian globular cytochrome b(5) fused to Escherichia coli alkaline phosphatase signal sequence (SS) was used as a model probe to investigate the influence of substituting each one of the standard 20 amino acids at its N-terminus on the Sec-dependent export of the precursor to the periplasmic space of E. coli. Substituting the native Met(+1) of the passenger protein flanking the SS with any one of the remaining 19 amino acids introduced significant changes in the export of cytochrome b(5) without jamming the Sec-dependent translocon.

Periplasmically-exported lupanine hydroxylase undergoes transition from soluble to functional inclusion bodies in Escherichia coli.

Pseudomonas lupanine hydroxylase is a periplasmic-localised, two domain quinocytochrome c enzyme. It requires numerous post-translocation modifications involving signal peptide processing, disulphide bridge formation and, heme linkage in the carboxy-terminal cytochrome c domain to eventually generate a Ca(2+)-bound quino-c hemoprotein that hydroxylates the plant alkaloid, lupanine. An exported, functional recombinant enzyme was generated in Escherichia coli by co-expression with cytochrome c maturation factors.

Signal peptidase I-mediated processing of an engineered mammalian cytochrome b(5) precursor is an exocytoplasmic post-translocational event in Escherichia coli.

Proteins destined for translocation across the prokaryotic cytoplasmic membrane are synthesized as precursors carrying transient N-terminal extensions known as signal sequences. They facilitate initial engagement of precursor proteins with the sec-dependent translocase to initiate active threading of the polypeptide across the membrane. The translocated precursor is then processed by a transcytoplasmic signal peptidase anchored to the inner membrane.