Electronic structure studies of the adenosylcobalamin cofactor in glutamate mutase

Glutamate mutase (GM) is a cobalamin-dependent enzyme that catalyzes the reversible interconversion of L-glutamate and L-threo-3-methylaspartate via a radical-based mechanism. To initiate catalysis, the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor's Co-C bond is cleaved homolytically to generate an adenosyl radical and Co2+ Cbl. In this work, we employed a combination of spectroscopic and computational tools to evaluate possible mechanisms by which the Co-C bond is activated for homolysis. Minimal perturbations to the electronic absorption (Abs), circular dichroism (CD), and magnetic CD (MCD) spectra of AdoCbl are observed upon formation of holoenzyme, even in the presence of substrate (or a substrate analogue), indicating that destabilization of the Co3+ Cbl "ground state" is an unlikely mechanism for Co-C bond activation. In contrast, striking alterations are observed in the spectroscopic data of the post-homolysis product Co2+ Cbl when bound to glutamate mutase in the presence of substrate (or a substrate analogue) as compared to unbound Co2+ Cbl. These enzymatic perturbations appear to most strongly affect the metal-to-ligand charge-transfer transitions of Co2+ Cbl, suggesting that the cofactor/active-site interactions give rise to a fairly uniform stabilization of the Co 3d orbitals. Remarkable similarities between the results obtained in this study and those reported previously for the related Cbl-dependent isomerase methylmalonyl-CoA mutase indicate that a common mechanism by which the cofactor's Co-C bond is activated for homolytic cleavage may be operative for all base-off/His-on Cbl-dependent isomerases.     

Redox regulation and reaction mechanism of human cystathionine-beta-synthase: a PLP-dependent hemesensor protein

Cystathionine beta-synthase in mammals lies at a pivotal crossroad in methionine metabolism directing flux toward cysteine synthesis and catabolism. The enzyme exhibits a modular organization and complex regulation. It catalyzes the beta-replacement of the hydroxyl group of serine with the thiolate of homocysteine and is unique in being the only known pyridoxal phosphate-dependent enzyme that also contains heme b as a cofactor. The heme functions as a sensor and modulates enzyme activity in response to redox change and to CO binding. Mutations in this enzyme are the single most common cause of hereditary hyperhomocysteinemia. Elucidation of the crystal structure of a truncated and highly active form of the human enzyme containing the heme- and pyridoxal phosphate binding domains has afforded a structural perspective on mechanistic and mutation analysis studies. The C-terminal regulatory domain containing two CBS motifs exerts intrasteric regulation and binds the allosteric activator, S-adenosylmethionine. Studies with mammalian cells in culture as well as with animal models have unraveled multiple layers of regulation of cystathionine beta-synthase in response to redox perturbations and reveal the important role of this enzyme in glutathione-dependent redox homestasis. This review discusses the recent advances in our understanding of the structure, mechanism, and regulation of cystathionine beta-synthase from the perspective of its physiological function, focusing on the clinically relevant human enzyme.     

A B12-responsive internal ribosome entry site (IRES) element in human methionine synthase

Regulation of homocysteine, a sulfur-containing amino acid that is a risk factor for cardiovascular diseases, is poorly understood. Methionine synthase (MS) is a key enzyme that clears intracellular homocysteine, and its activity is induced by its cofactor, vitamin B12, at a translational level. In this study, we demonstrate that translation of MS, which has a long and highly structured 5'-untranslated region, is initiated from an internal ribosome entry site (IRES), which is modulated by B12. The minimal IRES element spans 71 bases immediately upstream of the initiation codon. Electrophoretic mobility shift analysis reveals the presence of a B12 -dependent protein-RNA complex and suggests the possibility that B12-dependent increase of IRES efficiency is mediated via a protein. Modulation of the IRES-dependent translation of an essential gene by the cofactor of the encoded enzyme represents a novel example of a gene-nutrient interaction.     

Importance of photoperiods in the regulation of ovarian activities in indian major carp Catla catla in an annual cycle

The present study attempted for the first time to explore the importance of photoperiod in the regulation of seasonal ovarian functions in any subtropical major carp. Adult Indian major carp Catla catla were transferred to a long photoperiod (LP; LD 16:8) or a short photoperiod (SP; LD 8:16) for 30 days on 4 dates corresponding to the beginnings of 4 reproductive phases in an annual cycle, and responsiveness of the ovary was evaluated by comparison with the gonadal weight (I(G)), relative number of developing oocytes, serum levels of vitellogenin, and the activity of 2 important steroidogenic enzymes, that is, Delta(5)3beta-hydroxysteroid dehydrogenase and 17.beta-hydroxysteroid dehydrogenase, in the ovary of fish in a natural photoperiod. Exposure of fish to LP during the preparatory phase (February-March) resulted in a significant (p < 0.001) increase in the values of vitellogenin and in the activity of both the steroidogenic enzymes but not in the ovarian weight and in the relative number of different stages of oocytes. A more stimulatory influence of LP was noted during the prespawning phase (April-May), when precocious maturation of ovary was evident from a significant (p < 0.001) rise in the values of each studied features of ovarian functions. However, no ovarian response was found when the fish were transferred to LP during the spawning (July-August) and the postspawning (September-October) phases. On the other hand, the SP was found to have an inhibitory influence on ovarian growth and maturation during the prespawning and the spawning phases or to have no influences on ovarian functions during the preparatory and the postspawning phases of an annual cycle. The results of our study provide the first evidence that photoperiod per se plays an important role in the seasonal maturation of ovary in a subtropical freshwater major carp.     

Stopped-flow kinetic analysis of the reaction catalyzed by the full-length yeast cystathionine beta-synthase

Cystathionine beta-synthase found in yeast catalyzes a pyridoxal phosphate-dependent condensation of homocysteine and serine to form cystathionine. Unlike the homologous mammalian enzymes, yeast cystathionine beta-synthase lacks a second cofactor, heme, which facilitates detailed kinetic studies of the enzyme because the different pyridoxal phosphate-bound intermediates can be followed by their characteristic absorption spectra. We conducted a rapid reaction kinetic analysis of the full-length yeast enzyme in the forward and reverse directions. In the forward direction, we observed formation of the external aldimine of serine (14 mm(-1) s(-1)) and the aminoacrylate intermediate (15 s(-1)). Homocysteine binds to the aminoacrylate with a bimolecular rate constant of 35 mm(-1) s(-1) and rapidly converts to cystathionine (180 s(-1)), leading to the accumulation of a 420 nm absorbing species, which has been assigned as the external aldimine of cystathionine. Release of cystathionine is slow (k = 2.3 s(-1)), which is similar to k(cat) (1.7 s(-1)) at 15 degrees C, consistent with this being a rate-determining step. In the reverse direction, cystathionine binds to the enzyme with a bimolecular rate constant of 1.5 mm(-1) s(-1) and is rapidly converted to the aminoacrylate without accumulation of the external aldimine. The kinetic behavior of the full-length enzyme shows notable differences from that reported for a truncated form of the enzyme lacking the C-terminal third of the protein (Jhee, K. H., Niks, D., McPhie, P., Dunn, M. F., and Miles, E. W. (2001) Biochemistry 40, 10873-10880).     

Transient Kinetic Analysis of Hydrogen Sulfide Oxidation Catalyzed by Human Sulfide Quinone Oxidoreductase

The first step in the mitochondrial sulfide oxidation pathway is catalyzed by sulfide quinone oxidoreductase (SQR), which belongs to the family of flavoprotein disulfide oxidoreductases. During the catalytic cycle, the flavin cofactor is intermittently reduced by sulfide and oxidized by ubiquinone, linking H₂S oxidation to the electron transfer chain and to energy metabolism. Human SQR can use multiple thiophilic acceptors, including sulfide, sulfite, and glutathione, to form as products, hydrodisulfide, thiosulfate, and glutathione persulfide, respectively. In this study, we have used transient kinetics to examine the mechanism of the flavin reductive half-reaction and have determined the redox potential of the bound flavin to be −123 ± 7 mV. We observe formation of an unusually intense charge-transfer (CT) complex when the enzyme is exposed to sulfide and unexpectedly, when it is exposed to sulfite. In the canonical reaction, sulfide serves as the sulfur donor and sulfite serves as the acceptor, forming thiosulfate. We show that thiosulfate is also formed when sulfide is added to the sulfite-induced CT intermediate, representing a new mechanism for thiosulfate formation. The CT complex is formed at a kinetically competent rate by reaction with sulfide but not with sulfite. Our study indicates that sulfide addition to the active site disulfide is preferred under normal turnover conditions. However, under pathological conditions when sulfite concentrations are high, sulfite could compete with sulfide for addition to the active site disulfide, leading to attenuation of SQR activity and to an alternate route for thiosulfate formation.     

Psychrotolerant antifungal Streptomyces isolated from Tawang, India and the shift in chitinase gene family

A total of 210 Streptomyces were isolated from the soil samples of Tawang, India where temperature varied from 5 °C during daytime to ?2 °C during the night. Based on antifungal activity, a total of 33 strains, putatively Streptomyces spp., were selected. Optimal growth temperature for the 33 strains was 16 °C, with growth occurring down to 6 °C but not above 30 °C. Phylogenetic analysis based on 16S rDNA sequences revealed the taxonomic affiliation of the 33 strains as species of Streptomyces. To examine the relatedness of the chitinase genes from six strong antifungal Streptomyces strains, a phylogenetic tree was constructed using the catalytic domain nucleotide sequences and resulted in seven distinct monophyletic groups. A quantitative PCR study for chitinase expressing ability revealed that of the six antifungal strains tested, the strain Streptomyces roseochromogenus TSR12 was the most active producer of family 18 chitinase genes. Streptomyces strains with enhanced inhibitory potential usually encode a family 19 chitinase gene; however, our present study did not show expression of this family in the six strains tested.     

Comparative genomics of cell envelope components in mycobacteria

Mycobacterial cell envelope components have been a major focus of research due to their unique features that confer intrinsic resistance to antibiotics and chemicals apart from serving as a low-permeability barrier. The complex lipids secreted by Mycobacteria are known to evoke/repress host-immune response and thus contribute to its pathogenicity. This study focuses on the comparative genomics of the biosynthetic machinery of cell wall components across 21-mycobacterial genomes available in GenBank release 179.0. An insight into survival in varied environments could be attributed to its variation in the biosynthetic machinery. Gene-specific motifs like 'DLLAQPTPAW' of ufaA1 gene, novel functional linkages such as involvement of Rv0227c in mycolate biosynthesis; Rv2613c in LAM biosynthesis and Rv1209 in arabinogalactan peptidoglycan biosynthesis were detected in this study. These predictions correlate well with the available mutant and coexpression data from TBDB. It also helped to arrive at a minimal functional gene set for these biosynthetic pathways that complements findings using TraSH.     

Loss of allostery and coenzyme B12 delivery by a pathogenic mutation in adenosyltransferase

ATP-dependent cob(I)alamin adenosyltransferase (ATR) is a bifunctional protein: an enzyme that catalyzes the adenosylation of cob(I)alamin and an escort that delivers the product, adenosylcobalamin (AdoCbl or coenzyme B(12)), to methylmalonyl-CoA mutase (MCM), resulting in holoenzyme formation. Failure to assemble holo-MCM leads to methylmalonic aciduria. We have previously demonstrated that only 2 equiv of AdoCbl bind per homotrimer of ATR and that binding of ATP to the vacant active site triggers ejection of 1 equiv of AdoCbl from an adjacent site. In this study, we have mimicked in the Methylobacterium extorquens ATR, a C-terminal truncation mutation, D180X, described in a patient with methylmalonic aciduria, and characterized the associated biochemical penalties. We demonstrate that while k(cat) and K(M)(Cob(I)) for D180X ATR are only modestly decreased (by 3- and 2-fold, respectively), affinity for the product, AdoCbl, is significantly diminished (400-fold), and the negative cooperativity associated with its binding is lost. We also demonstrate that the D180X mutation corrupts ATP-dependent cofactor ejection, which leads to transfer of AdoCbl from wild-type ATR to MCM. These results suggest that the pathogenicity of the corresponding human truncation mutant results from its inability to sequester AdoCbl for direct transfer to MCM. Instead, cofactor release into solution is predicted to reduce the capacity for holo-MCM formation, leading to disease.