Effects of polynitrogen compounds on the activity of recombinant human HIF-1α prolyl hydroxylase 3 in E. coli.

Hypoxia inducible factor 1α (HIF-1α) becomes an important regulation factor within the histiocyte when it is under the hypoxia condition. Recently, prolyl hydroxylases (PHDs) have been identified to inactivation HIF-lα by hydroxylation. In this study, polynitrogen compounds were screened as HIF-1α PHD3 inhibitors. The coding region of human PHD3 DNA was optimized by using synonymous codons according to the code bias of Escherichia coli. Soluble and active human PHD3 was expressed in the E. coli with a Trx fusion tag under a lower induction temperature of 25°C. Mass spectrometry analysis of the resultant peptide product indicated a mass increase of 16 daltons, consistent with hydroxylation of the proline residue in the HIF-1α (556-574) peptide substrate. Polynitrogen compounds (1-4) inhibited the enzymatic hydroxylation of HIF-1α peptide in a concentration-dependent manner, and the apparent IC(50) values were 29.5, 16.0, 12.8 and 60.4 µM respectively. Double reciprocal (1/V versus 1/[HIF-1α peptide]) plots showed that these compounds are noncompetitive inhibitors of the hydroxylation by recombinant human PHD3 with K(i) values of 67.0, 25.3, 67.3, and 82.1 µM respectively. On the other hand, the metal complexes of these polynitrogen compounds (1-4) cannot inhibit the catalytical activity of PHD3. We hypothesized that the inhibitory mechanism of PHD3 activity by polynitrogen compounds is due to their binding to iron to form stable coordination complexes. Our results in this study indicated that polynitrogen compounds (1-4) could be potential inhibitors of PHD3 to regulate the transcriptional activity of HIF-1α.

New insights into the mechanism of arsenite methylation with the recombinant human arsenic (+3) methyltransferase (hAS3MT).

The catalytic mechanism of the recombinant human arsenic (+3) methyltransferase (hAS3MT) was studied using kinetics, initial velocity and spectroscopy. The production and the distribution of methylated arsenicals changed with various concentrations of arsenite/S-adenosyl-L-methionine (SAM)/thiols, enzyme contents, and incubation times. These results suggest a sequential methylation of arsenite to monomethylated arsenicals (MMA) and dimethylated arsenicals (DMA). In addition, competition exists between the two reactions. hAS3MT showed the greatest activity at pH 8.5 with glutathione (GSH) as the reductant. This might indicate that a balance between the deprotonation and protonation of sulfhydryl groups is required. Initial velocity studies illuminate an ordered sequence for the binding of SAM and arsenite to the hAS3MT; while GSH should probably be placed either as the first reactant or as a reactant combining with the enzyme only after products have been released. The interactions between substrate/cofactors and the hAS3MT were first monitored by UV-visible and circular dichroism spectroscopy. It revealed that arsenite and SAM combined with the hAS3MT before reaction started; whereas, no interactions between GSH and the hAS3MT were detected. Integrating the results from kinetics, initial velocity and spectroscopy studies, an ordered mechanism are originally attained, with the SAM as the first reactant that adds to the hAS3MT and arsenite as the second one. Arsenite is successively methylated reductively, rather than a stepwise oxidative methylation. GSH should combine with the hAS3MT after the methylation to reduce the disulfide bond formed during the catalytic cycle in the hAS3MT to resume the active form of the enzyme.

Study on a hidden protein-DNA binding in salmon sperm DNA sample by dynamic kinetic capillary isoelectric focusing.

Nuclease P1 is an important enzyme that hydrolyzes RNA or single-stranded DNA into nucleotides, and complete digestion is an essential basis for assays based on this enzyme. To digest a doubled-stranded DNA, the enzyme is usually combined with heat denaturing, which breaks doubled-stranded DNA into single strands. This paper presents an un-expected phenomenon that nuclease P1, in combination with heat denaturing, fails to completely digest a DNA sample extracted from salmon sperm. Under the experimental conditions used, at which nuclease P1 can completely digest calf thymus DNA, the digestion yield of salmon sperm DNA was only 89.5%. Spectrometric measurement indicated that a total protein of 4.7% is present in the DNA sample. To explain the reason for this phenomenon, the dynamic kinetic capillary isoelectric focusing (DK-CIEF) approach proposed previously, which allows for the discrimination of different types of protein-DNA interactions and the measurement of the individual dissociation rate constants, was modified and applied to examine possible protein-DNA interactions involved. It was found that a non-specific DNA-protein binding occurs in the sample, the dissociation rate constant for which was measured to be 7.05+/-0.83x10(-3) s(-1). The formation of DNA-protein complex was suggested to be the main reason for the incomplete digestion of the DNA sample. The modified DK-CIEF approach can be applied as general DNA samples, with the advantages of fast speed and low sample consumption.

Structure-function roles of four cysteine residues in the human arsenic (+3 oxidation state) methyltransferase (hAS3MT) by site-directed mutagenesis.

Cysteine (Cys) residues are often crucial to the function and structure of proteins. Cys157 and Cys207 in recombinant mouse arsenic (+3 oxidation state) methyltransferase (AS3MT) are shown to be related to enzyme activity and considered to be the catalytic sites. The roles of some conserved Cys residues in the N-terminal region of the rat AS3MT also have been examined. However, little is known about the roles of the Cys residues in the middle region. The metabolism of inorganic arsenic in human is different from rat and mouse in some aspects though the AS3MT has a high degree of similarity in these species. In order to determine whether the Cys156 and Cys206 (corresponding to the catalytic sites, Cys157 and Cys207 in the mouse AS3MT) in the hAS3MT act as the catalytic sites and to study the roles of the Cys residues (Cys226 and Cys250) near the catalytic center in the middle region, we designed and prepared four mutants (C156S, C206S, C226S, and C250S) in which one Cys residue replaced by serine by PCR-based site-directed mutagenesis. The native form and cysteine/serine mutants were assayed for enzyme activity, free thiols, and the secondary structures by circular dichroism and Fourier transform infrared. Our data show that, besides C156S and C206S, C250S is another potential important site. C226S seems to have the same action as the wild-type hAS3MT with the consistent K(M) and V(max) values. Meanwhile, selenium can also inhibit the methylation of inorganic arsenic by C226S. All the mutants except C226S are calculated to have dramatic changes in the secondary structures. Cys250 might form an intramolecular disulfide bond with another Cys residue. These findings demonstrate that Cys residues at positions 156, 206, and 250 play important roles in the enzymatic function and structure of the hAS3MT.