Mixed regioselectivity in the Arg-177 mutants of Corynebacterium diphtheriae heme oxygenase as a consequence of in-plane heme disorder

It has been reported that the R183E and R183D mutants of rat heme oxygenase-1 (r-HO-1) produce approximately 30% delta-biliverdin [Zhou, H., et al. (2000) J. Am. Chem. Soc. 122, 8311-8312]. Two plausible mechanisms were proposed to explain the observations. (a) Electrostatic repulsion between E183 (D183) and one of the heme propionates forces the heme to rotate, thereby placing the delta-meso carbon in a position that is susceptible to oxidation. (b) Rearrangement of the distal pocket structure is triggered by the formation of a hydrogen bond between E183 (D183) and K179. A change in the pK(a) for the Fe(III)-H(2)O to Fe(III)-OH transition of the mutants was interpreted to be consistent with rearrangement of the hydrogen bond network in the distal pocket. The large similarities between the high-frequency portion of the (1)H NMR spectra corresponding to the wild type and R183E and R183D mutants were interpreted to indicate that the heme in the mutants is not rotated to a significant extent. We have re-examined this issue by studying the corresponding R177 mutants in heme oxygenase from Corynebacterium diphtheriae (cd-HO). Replacing R177 with E or D results in the formation of approximately 55% alpha- and 45% delta-biliverdin, whereas the R177A mutant retains alpha-regioselectivity. In addition, the K13N/Y130F/R177A triple mutant catalyzed the formation of 60% delta- and 40% alpha-biliverdin, while single mutants K13N and Y130F did not appreciably change the regioselectivity of the reaction. The pK(a) of the Fe(III)-H(2)O to Fe(III)-OH transition in wild-type cd-HO is 9.1, and those of the R177E, R177D, R177A, and K13N/Y130F/R177A mutants are 9.4, 9.5, 9.2, and 8.0, respectively. Thus, no obvious correlation exists between the changes in pK(a) and the altered regioselectivity. NMR spectroscopic studies conducted with the R177D and R177E mutants of cd-HO revealed the presence of three heme isomers: a major (M) and a minor (m) heme orientational isomer related by a 180 degrees rotation about the alpha-gamma meso axis and an alternative seating (m') which is related to m by an 85 degrees in-plane rotation of the macrocycle. The in-plane rotation of m to acquire conformation m' is triggered by electrostatic repulsion between the side chains of D or E at position 177 and heme propionate-6. As a consequence, the delta-meso carbon in m' is placed in the position occupied by the alpha-meso carbon in m, where it is susceptible to hydroxylation and subsequent formation of delta-biliverdin.     

Crystal structures of the NO- and CO-bound heme oxygenase from Neisseriae meningitidis. Implications for O2 activation

Heme oxygenases catalyze the oxidation of heme to biliverdin, carbon monoxide, and free iron while playing a critical role in mammalian heme homeostasis. Pathogenic bacteria such as Neisseriae meningitidis also produce heme oxygenase as part of a mechanism to mine host iron. The key step in heme oxidation is the regioselective oxidation of the heme alpha-meso-carbon by an activated Fe(III)-OOH complex. The structures of various diatomic ligands bound to the heme iron can mimic the dioxygen complex and provide important insights on the mechanism of O2 activation. Here we report the crystal structures of N. meningitidis heme oxygenase (nm-HO) in the Fe(II), Fe(II)-CO, and Fe(II)-NO states and compare these to the NO complex of human heme oxygenase-1 (Lad, L., Wang, J., Li, H., Friedman, J., Bhaskar, B., Ortiz de Montellano, P. R., and Poulos, T. L. (2003) J. Mol. Biol. 330, 527-538). Coordination of NO or CO results in a reorientation of Arg-77 that enables Arg-77 to participate in an active site H-bonded network involving a series of water molecules. One of these water molecules directly H-bonds to the Fe(II)-linked ligand and very likely serves as the proton source required for oxygen activation. Although the active site residues differ between nm-HO and human HO-1, the close similarity in the H-bonded water network suggests a common mechanism shared by all heme oxygenases.     

Structural characterizations of nonpeptidic thiadiazole inhibitors of matrix metalloproteinases reveal the basis for stromelysin selectivity.

The binding of two 5-substituted-1,3,4-thiadiazole-2-thione inhibitors to the matrix metalloproteinase stromelysin (MMP-3) have been characterized by protein crystallography. Both inhibitors coordinate to the catalytic zinc cation via an exocyclic sulfur and lay in an unusual position across the unprimed (P1-P3) side of the proteinase active site. Nitrogen atoms in the thiadiazole moiety make specific hydrogen bond interactions with enzyme structural elements that are conserved across all enzymes in the matrix metalloproteinase class. Strong hydrophobic interactions between the inhibitors and the side chain of tyrosine-155 appear to be responsible for the very high selectivity of these inhibitors for stromelysin. In these enzyme/inhibitor complexes, the S1'' enzyme subsite is unoccupied. A conformational rearrangement of the catalytic domain occurs that reveals an inherent flexibility of the substrate binding region leading to speculation about a possible mechanism for modulation of stromelysin activity and selectivity.     

JAK protein tyrosine kinases: their role in cytokine signalling

Protein tyrosine kinases (PTKs) are integral components of the cellular machinery that mediates the transduction and/or processing of many extra- and intracellular signals. Members of the JAK family of intracellular PTKs (JAK1, JAK2 and TYK2) are characterized by the possession of a PTK-related domain and five additional homology domains, in addition to a classical PTK domain. An important breakthrough in the understanding of JAK kinases function(s) has come from the recent observations that many cytokine receptors compensate for their lack of a PTK domain by utilizing members of the JAK family for signal transduction.     

Additional studies on pregnancy termination and inhibition of the monkey corpus luteum with 5-oxa-17-phenyl-18,19,20-trinor-PGF1α methyl ester and structurally related prostaglandins

Oral administration of 5-oxa-17-phenyl-18,19,20-trinor-PGF_1α methyl ester (PGF-analog) resulted in a consistent and dose-dependent inhibition of corpus luteum progesterone production in nonpregnant rhesus monkeys concomitantly treated with human chorionic gonadotropin. Similarly, vaginal suppositories containing PGF-analog also inhibited the monkey corpus luteum. Side effects by the oral route of administration were minimal, whereas side effects of following vaginal treatment with PGF-analog were higher. Five prostaglandings with structural similarity to PGF-analog were studied for their ability to inhibit the monkey corpus luteum, but none showed an advantage over the parent molecule. PGF-analog did not synergize with 9-deoxo-16,16-dimethyl-9-methylene-PGE₂ for the inhibition of the monkey corpus luteum, nor did it synergize with (15S)-15-methyl-PGF₂α methyl ester for the interruption of early pregnancy in the monkey. 9-Deoxo-9-methylene-5-oxa-17-phenyl-18,19,20-trinor-PGE₁ methyl ester did not terminate early gestation in the monkey at doses of 8 or 24 mg.     

Identification of the proximal ligand His-20 in heme oxygenase (Hmu O) from Corynebacterium diphtheriae. Oxidative cleavage of the heme macrocycle does not require the proximal histidine

The coordination and spin-state of the Corynebacterium diphtheriae heme oxygenase (Hmu O) and the proximal Hmu O H20A mutant have been characterized by UV-visible and resonance Raman (RR) spectrophotometry. At neutral pH the ferric heme-Hmu O complex is a mixture of six-coordinate high spin and six-coordinate low spin species. Changes in the UV-visible and high frequency RR spectra are observed as a function of pH and temperature, with the six-coordinate high spin species being converted to six-coordinate low spin. The low frequency region of the ferrous RR spectrum identified the proximal ligand to the heme as a neutral imidazole with a Fe-His stretching mode at 222 cm(-1). The RR characterization of the heme-CO complex in wt-Hmu O confirms that the proximal imidazole is neither ionized or strongly hydrogen-bonded. Based on sequence identity with the mammalian enzymes the proximal ligand in HO-1 (His-25) and HO-2 (His-45) is conserved (His-20) in the bacterial enzyme. Site-specific mutagenesis identified His-20 as the proximal mutant based on electronic and resonance Raman spectrophotometric analysis. Titration of the heme-Hmu O complex with imidazole restored full catalytic activity to the enzyme, and the coordination of imidazole to the heme was confirmed by RR. However, in the absence of imidazole, the H20A Hmu O mutant was found to catalyze the initial alpha-meso-hydroxylation of the heme. The product of the aerobic reaction was determined to be ferrous verdoheme. Hydrolytic conversion of the verdoheme product to biliverdin concluded that oxidative cleavage of the porphyrin macrocycle was specific for the alpha-meso-carbon. The present data show that, in marked contrast to the human HO-1, the proximal ligand is not essential for the initial alpha-meso-hydroxylation of heme in the C. diphtheriae heme oxygenase-catalyzed reaction.     

Insulin resistance in adolescents with Down syndrome: a cross-sectional study

Background

The prevalence of diabetes mellitus is higher in individuals with Down syndrome (DS) than in the general population; it may be due to the high prevalence of obesity presented by many of them. The aim of this study was to evaluate the insulin resistance (IR) using the HOMA (Homeostasis Model Assessment) method, in DS adolescents, describing it according to the sex, body mass index (BMI) and pubertal development.

Methods

15 adolescents with DS (8 males and 7 females) were studied, aged 10 to 18 years, without history of disease or use of medication that could change the suggested laboratory evaluation. On physical examination, the pubertal signs, acanthosis nigricans (AN), weight and height were evaluated. Fasting plasma glucose and insulin were analysed by the colorimetric method and RIA-kit LINCO, respectively. IR was calculated using the HOMA method. The patients were grouped into obese, overweight and normal, according to their BMI percentiles. The EPIINFO 2004 software was used to calculate the BMI, its percentile and Z score.

Results

Five patients were adults (Tanner V or presence of menarche), 9 pubertal (Tanner II – IV) and 1 prepubertal (Tanner I). No one had AN. Two were obese, 4 overweight and 9 normal. Considering the total number of patients, HOMA was 1.7 ± 1.0, insulin 9.3 ± 4.8 μU/ml and glucose 74.4 ± 14.8 mg/dl. The HOMA values were 2.0 ± 1.0 in females and 1.5 ± 1.0 in males. Considering the nutritional classification, the values of HOMA and insulin were: HOMA: 3.3 ± 0.6, 2.0 ± 1.1 and 1.3 ± 0.6, and insulin: 18.15 ± 1.6 μU/ml, 10.3 ± 3.5 μU/ml and 6.8 ± 2.8 μU/ml, in the obese, overweight and normal groups respectively. Considering puberty, the values of HOMA and insulin were: HOMA: 2.5 ± 1.3, 1.4 ± 0.6 and 0.8 ± 0.0, and insulin: 13.0 ± 5.8 μU/ml, 7.8 ± 2.9 μU/ml and 4.0 ± 0.0 μU/ml, in the adult, pubertal and prepubertal groups respectively.

Conclusion

The obese and overweight, female and adult patients showed the highest values of HOMA and insulin.     

The ShuS protein of Shigella dysenteriae is a heme-sequestering protein that also binds DNA

The ability of Shigella dysenteriae to utilize heme as an iron source is dependent on the iron-regulated expression of a number of genes including the outermembrane receptor ShuA and the cytoplasmic protein ShuS. The ShuS protein has no sequence homology with any proteins of known function and its role in heme acquisition has not been determined. In this paper we describe the purification and characterization of ShuS. The soluble oligomeric protein (650 kDa) is composed of a single type of subunit with a molecular mass of 37 kDa and binds one heme per monomer (Kd = 13 microM). In addition, the ShuS protein was shown to nonspecifically bind double-stranded DNA. It appears, therefore, that ShuS may function as both a heme storage protein, during periods of active heme transport, and as a DNA binding protein to protect the DNA from any ensuing heme mediated oxidative damage.