No abstract

P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redoxpartner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate.

The last gene in the nuo operon of Escherichia coli, nuoN, encodes a membrane-bound subunit of Complex I (NADH:ubiquinone oxidoreductase). In this report, the gene for subunit N was disrupted by a 163 bp deletion in the chromosome, resulting in the loss of Complex I function, as measured by deamino-NADH oxidase activity. This activity could be recovered after transformation of the mutant strain by a plasmid that contains the previously identified nuoN gene and the upstream intergenic region between nuoM and nuoN. Mutagenesis of the first ATG downstream of nuoM led to a loss of function, indicating that this is the likely initiation codon for nuoN, and predicting a protein of 485 amino acids and 52 044 Da. Thirty site-specific mutations in nuoN at 19 different positions were constructed in a vector that expresses the full-length subunit N with both an octahistidine tag and an HA epitope tag at the carboxyl terminus. Highly conserved charged and aromatic residues were selected for mutagenesis, as well as a substitution that occurs as a secondary mutation in Leber's hereditary optic neuropathy (LHON). Membranes from the mutant strains were tested for production of subunit N by immunoblots and for NADH-linked activities. Mutants with substitutions at six different positions (K158, K217, H224, K247, Y300, and K395) had rates of deamino-NADH oxidase activity that were no more than 50% of that of the wild type and had reduced rates of proton translocation. These mutants also showed enhanced inhibition by decylubiquinone, indicating that subunit N interacts with quinones. The mutation associated with LHON, G391S, had little effect on these functions.

Mutagenesis studies indicated that the three cytoplasmic cysteines of the influenza virus A/Japan/305/57 hemagglutinin (HA) are all palmitylated, but to an unequal extent. Replacement of all three cysteines abolished palmitylation, but affected neither HA biosynthesis nor function. Palmitate was not required for HA to be incorporated into virions.

The relationship of function to structure of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) has been examined by means of sequence alignment and site-directed mutagenesis. Comparison has been made between the sequence of P450arom and the two soluble bacterial cytochrome P450 isoforms, whose three-dimensional structure has been determined (P450BM3 and P450cam). From this comparison, it appears that although there is a similarity of overall structure in cytochromes P450, there is enough significant difference in the regions involved in substrate recognition and substrate binding that residues believed to be involved, even in the known structures, must be tested. With this in mind, we have generated a detailed alignment of P450arom, including the definition of putative alpha-helices and beta-sheets based on comparison of the alignments of P450BM3 and P450cam, generated from their three-dimensional structure, and have made mutations in regions we believe to be involved in substrate recognition at the solvent surface and orientation in the heme pocket. We have mutated F116 and F134 to determine if they are present in the heme pocket, and Q225 and L228 to determine if they are a part of the substrate recognition loop. Although F116E is essentially inactive and may be a folding mutant or may inhibit reductase binding, F134E is more active than the wild type and may be located in the heme pocket facilitating the hydrogen abstraction from C2 of androstenedione. Mutations at Q225 and L228 also result in the anticipated changes in the apparent Km and maximum velocity.(ABSTRACT TRUNCATED AT 250 WORDS)