[Antley-Bixler syndrome or POR deficiency?]. / Antley-Bixleruv syndrom nebo POR deficience?

Antley-Bixler syndrome (ABS) is a rare congenital disorder characterized by numerous craniofacial, skeletal and, in some cases, urogenital abnormalities resulting from disordered steroidogenesis. Known genetic causes in sporadic cases of ABS include dominant mutations in the fibroblast growth factor 2 receptor gene (FGFR2). Recent research shows surprisingly that symptoms of Antley-Bixler syndrome, combined with disordered steroidogenesis and urogenital anomalies, are caused by mutations in the POR gene that encodes NADPH-cytochrome P450 oxidoreductase (CYPOR). CYPOR is a four domain-containing monomeric flavoprotein that contains two flavins, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), a binding site for NADPH, and the N-terminal sequence of 25 amino acids which determines the microsomal localization of the protein. CYPOR is the electron donor to microsomally localized cytochromes P450 that participate in xenobiotic metabolism and steroidogenesis. Mutations in the POR gene lead to apparent diminished activity of some P450 enzymes. Association of CYPOR with ABS discloses new facts about this disease and recent research shows that patients with ABS-like skeletal anomalies, but with mutations in the POR gene and disordered steroidogenesis, represent a new disorder called POR deficiency.

The structure and biochemistry of NADH-dependent cytochrome b5 reductase are now consistent.

Cytochrome b5 reductase (cb5r) (EC 1.6.6.2) catalyzes the reduction of two molecules of cytochrome b5 using NADH as the physiological electron donor. The structure of pig cb5r at 2.4 A resolution was previously reported in the literature, but it was inconsistent with the biochemistry; for example, K83 and C245 were both implicated in the mechanism, but were not located at the active site. To address this problem, we have determined the structures of cb5r from rat at 2.0 A resolution and in a complex with NAD+ at 2.3 A resolution. We found significant differences throughout the rat structure compared to that of pig, including the locations of the lysine and cysteine residues mentioned above. To test the structural models, we made single amino acid substitutions of this lysine and showed that all substitutions produced correctly folded proteins and exhibited normal flavin behavior. However, the apparent kcat(NADH) decreased, and the apparent K(m) for NADH increased; the K(m)'s for cytochrome b5 were unchanged relative to that of the wild type. The largest effect was for the glutamate-substituted protein, which was further characterized using a charge transfer assay and found to be less efficient at NADH utilization than the wild type. These results are consistent with a role for this lysine in stabilizing the NADH-bound form of cb5r. We have concluded that the pig structure was mistraced in several regions and have reinterpreted mutants in these regions that give rise to the hereditary disease methemoglobinemia.

Assimilatory nitrate reductase: lysine 741 participates in pyridine nucleotide binding via charge complementarity.

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b557-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by high plants. With a recombinant, histidine-tagged form of the spinach nitrate reductase flavin domain, site-directed mutagenesis has been utilized to examine the role of lysine 741 in binding the reducing substrate, NADH. Seven individual mutants, corresponding to K741R, K741H, K741A, K741E, K741M, K741Q, and K741P, have been engineered and six of the resulting proteins purified to homogeneity. With the exception of K741P, all the mutants were obtained as functional flavoproteins which retained FAD as the sole prosthetic group and exhibited spectroscopic properties comparable to those of the wild-type domain, indicating that the amino acid substitutions had no effect on FAD binding. In contrast, all the mutants were found to have altered NADH:ferricyanide reductase (NADH:FR) activity with mutations affecting both kcat and K(NADH)m, which decreased and increased, respectively. At pH 7.0, kcat decreased in the order WT > K741R > K741A > K741H > K741E > K741M > K741Q while K(NADH)m increased in the same order. The most efficient mutant, K741R, retained 80% of the wild-type NADH:FR activity, while in contrast the most inefficient mutant, K741Q, retained only 18% of the wild-type NADH:FR activity together with a 118-fold increased K(NADH)m. pH studies of K741H revealed that both kcat and K(NADH)m were pH-dependent, with enhanced activity observed at acidic pH. These results indicated that retention of a positively charged side chain at position 741 in the spinach nitrate reductase primary sequence is important for the efficient binding and subsequent oxidation of NADH and that the positively charged side chain enhances nucleotide binding via charge complementarity with the negatively charged pyrophosphate moiety.

Arginine 91 is not essential for flavin incorporation in hepatic cytochrome b(5) reductase.

Cytochrome b(5) reductase (cb5r) catalyzes the transfer of reducing equivalents from NADH to cytochrome b(5). Utilizing an efficient heterologous expression system that produces a histidine-tagged form of the hydrophilic, diaphorase domain of the enzyme, site-directed mutagenesis has been used to generate cb5r mutants with substitutions at position 91 in the primary sequence. Arginine 91 is an important residue in binding the FAD prosthetic group and part of a conserved "RxY(T)(S)xx(S)(N)" sequence motif that is omnipresent in the "ferredoxin:NADP(+) reductase" family of flavoproteins. Arginine 91 was replaced with K, L, A, P, D, Q, and H residues, respectively, and all the mutant proteins purified to homogeneity. Individual mutants were expressed with variable efficiency and all exhibited molecular masses of approximately 32 kDa. With the exception of R91H, all the mutants retained visible absorption spectra typical of a flavoprotein, the former being produced as an apoprotein. Visible absorption spectra of R91A, L, and P were red shifted with maxima at 458 nm, while CD spectra indicated an altered FAD environment for all the mutants except R91K. Fluorescence spectra showed a reduced degree of intrinsic flavin fluorescence quenching for the R91K, A, and P, mutants, while thermal stability studies suggested all the mutants, except R91K, were somewhat less stable than the wild-type domain. Initial-rate kinetic measurements demonstrated that the mutants exhibited decreased NADH:ferricyanide reductase activity with the R91P mutant retaining the lowest activity, corresponding to a k(cat) of 283 s(-1) and a K(NADH)(m) of 105 microM, when compared to the wild-type domain (k(cat) = 800 s(-1) K(NADH)(m) = 6 microM). These results demonstrate that R91 is not essential for FAD binding in cb5r; however, mutation of R91 perturbs the flavin environment and alters both diaphorase substrate recognition and utilization.