Comparative localization of cystathionine beta-synthase and cystathionine gamma-lyase in retina: differences between amphibians and mammals.

Hydrogen sulfide (H(2)S) is a gaseous neuromodulator that can be synthesized by the transsulfuration enzymes cystathionine beta-synthase (CBS) and cystathionine gamma-lyase (CGL). In this study we examined H(2)S as a potential neuromodulator in vertebrate retina. CBS-like immunoreactivity (LI) was found in somas in the inner nuclear layer and as punctate staining in the inner and outer plexiform layers in the salamander retina. CGL-LI was most clearly characterized in salamander, where it was localized in Müller cells. Western blots indicated proteins with the correct molecular weights for both enzymes in both species for liver and cerebellum. Correct molecular weight proteins were identified for both CGL and CBS in salamander retina. The CBS antiserum did not recognize the correct molecular weight protein in mouse retina but the CGL antiserum recognized the correct molecular weight protein for mouse retina. Enzyme assays indicated both CGL and CBS enzyme activity in all three tissues in the salamander. There was good CBS activity in the liver and cerebellum of the mouse but no activity in the retina. CGL activity was clearly present only in the mouse liver, with only trace activity in the cerebellum and retina. In conclusion, both CBS and CGL are present in the amphibian retina, which suggests either a potential role for H(2)S as a gaseous neuromodulator in both neurons and glia in the retina or a requirement for cysteine and glutathione synthesis via the transsulfuration pathway as a defense against oxidative stress.

Expression study of mutant cystathionine beta-synthase found in Japanese patients with homocystinuria.

Cystathionine beta-synthase (CBS) deficiency is the most common cause of homocystinuria. More than 130 pathogenic mutations, mostly in the Caucasian populations, have been described. Recently, our group reported a mutation analysis of Japanese homocystinuric patients. In the present paper, we report an expression study of several mutant CBS enzymes in Escherichia coli, i.e., R121H, G148R, G151R, S217F, H232D, R266G, 1591delTTCG, and K441X. All of the mutants except K441X exhibited severely decreased activity, and the capability to form tetramers of most mutants was severely impaired. The K441X mutant, on the other hand, exhibited relatively high activity (63% of the wild type activity). This was probably due to two factors. First, the high abundance of the full-length CBS protein, a likely K441Q mutant, which was produced through suppression of the amber termination codon by glutamine tRNA in E. coli. And second, the presence of a C-terminally truncated protein, which was previously shown to be constitutively activated. Patient-derived lymphocytes, however, showed no detectable CBS subunits. As previously hypothesized, the increased aggregation of mutant CBS subunits might be a common pathogenic mechanism in CBS deficiency.

Inhibitor binding at the protein interface in crystals of a HIV-1 protease complex.

Depending on the excess of ligand used for complex formation, the HIV-1 protease complexed with a novel phenylnorstatine inhibitor forms crystals of either hexagonal (P6(1)) or orthorhombic (P2(1)2(1)2(1)) symmetry. The orthorhombic form shows an unusual complexity of crystal packing: in addition to one inhibitor molecule that is bound to the enzyme active site, the second inhibitor molecule is bound as an outer ligand at the protein interface. Binding of the outer ligand apparently increases the crystal-quality parameters so that the diffraction data allow solution of the structure of the complex at 1.03 A, the best resolution reported to date. The outer ligand interacts with all four surrounding HIV-1 protease molecules and has a bent conformation owing to its accommodation in the intermolecular space. The parameters of the solved structures of the orthorhombic and hexagonal forms are compared.

A phenylnorstatine inhibitor binding to HIV-1 protease: geometry, protonation, and subsite-pocket interactions analyzed at atomic resolution.

The X-ray structure of a complex of HIV-1 protease (PR) with a phenylnorstatine inhibitor Z-Pns-Phe-Glu-Glu-NH(2) has been determined at 1.03 A, the highest resolution so far reported for any HIV PR complex. The inhibitor shows subnanomolar K(i) values for both the wild-type PR and the variant representing one of the most common mutations linked to resistance development. The structure comprising the phenylnorstatine moiety of (2R,3S)-chirality displays a unique pattern of hydrogen bonding to the two catalytic aspartate residues. This high resolution makes it possible to assess the donor and acceptor relations of this hydrogen bonding and to indicate a proton shared by the two catalytic residues. A structural mechanism for the unimpaired inhibition of the protease Val82Ala mutant is also suggested, based on energy calculations and analyses.