Cystathionine beta-synthase is coordinately regulated with proliferation through a redox-sensitive mechanism in cultured human cells and Saccharomyces cerevisiae

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine with homocysteine to form cystathionine and occupies a crucial regulatory position between the methionine cycle and the biosynthesis of cysteine by transsulfuration. Analysis of CBS activity under a variety of growth conditions indicated that CBS is coordinately regulated with proliferation in both yeast and human cells. In batch cultures of Saccharomyces cerevisiae, maximal CBS activities were observed in the exponential phase of cells grown on glucose, while growth-arrested cultures or those growing non-fermentatively on ethanol or glycerol had approximately 3-fold less activity. CBS activity assays and Western blotting indicated that growth-specific regulation of CBS is evolutionarily conserved in a range of human cell lines. CBS activity was found to be maximal during proliferation and was reduced two- to five-fold when cells became quiescent at confluence. In cultured HepG2 cells, the human CBS gene is induced by serum and basic fibroblast growth factor and is downregulated, but not abolished, by contact inhibition, serum-starvation, nutrient depletion, or the induction of differentiation. Consequently, for certain cell types, CBS may represent a novel marker of both differentiation and proliferation. The intracellular level of the CBS regulator compound, S-adenosylmethionine, was found to reflect the proliferation status of both yeast and human cells, and as such, constitutes an additional mechanism for proliferation-specific regulation of human CBS. Our data indicates that screening compounds for the ability to affect transsulfuration in cultured cell models must take proliferation status into account to avoid masking regulatory interactions that may be of significance in vivo.     

Active cystathionine beta-synthase can be expressed in heme-free systems in the presence of metal-substituted porphyrins or a chemical chaperone

Cystathionine beta-synthase (CBS), a key enzyme in the metabolism of homocysteine, has previously been shown to require a heme co-factor for maximal activity. However, the biochemical function of the CBS heme is not well defined. Here, we show that expression of human CBS in heme-deficient strains of Saccharomyces cerevisiae and Escherichia coli results in production of an enzyme that is misfolded and degraded. Addition of exogenous heme, porphyrins with non-iron metal, or porphyrin lacking metal entirely produced stable and active CBS enzyme. Purification of recombinant CBS enzyme expressed in the presence of various metalloporphyrins confirmed that Mn(III) and Co(III) had 30-60% of the specific activity of Fe(III)-CBS, and still responded to allosteric activation by S-adenosyl-L-methionine. Treatment of S. cerevisiae with the chemical chaperone trimethylamine-N-oxide resulted in near complete restoration of function to human CBS produced in a heme-deficient strain. Taken together, these results suggest that porphyrin moiety of the heme plays a critical role in proper CBS folding and assembly, but that the metal ion is not essential for this function or for allosteric regulation by S-adenosyl-L-methionine.     

Human Polycomb 2 Protein Is a SUMO E3 Ligase and Alleviates Substrate-Induced Inhibition of Cystathionine β-Synthase Sumoylation

Human cystathionine β-synthase (CBS) catalyzes the first irreversible step in the transsulfuration pathway and commits homocysteine to the synthesis of cysteine. Mutations in CBS are the most common cause of severe hereditary hyperhomocysteinemia. A yeast two-hybrid approach to screen for proteins that interact with CBS had previously identified several components of the sumoylation pathway and resulted in the demonstration that CBS is a substrate for sumoylation. In this study, we demonstrate that sumoylation of CBS is enhanced in the presence of human polycomb group protein 2 (hPc2), an interacting partner that was identified in the initial yeast two-hybrid screen. When the substrates for CBS, homocysteine and serine for cystathionine generation and homocysteine and cysteine for H₂S generation, are added to the sumoylation mixture, they inhibit the sumoylation reaction, but only in the absence of hPc2. Similarly, the product of the CBS reaction, cystathionine, inhibits sumoylation in the absence of hPc2. Sumoylation in turn decreases CBS activity by ∼28% in the absence of hPc2 and by 70% in its presence. Based on these results, we conclude that hPc2 serves as a SUMO E3 ligase for CBS, increasing the efficiency of sumoylation. We also demonstrate that γ-cystathionase, the second enzyme in the transsulfuration pathway is a substrate for sumoylation under in vitro conditions. We speculate that the role of this modification may be for nuclear localization of the cysteine-generating pathway under conditions where nuclear glutathione demand is high.     

Dioxygen reactivity and heme redox potential of truncated human cystathionine beta-synthase

Cystathionine beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to cystathionine, which represents the committing step in the transsulfuration pathway. CBS is unique in being a pyridoxal phosphate-dependent enzyme that has a heme cofactor. The activity of CBS under in vitro conditions is responsive to the redox state of the heme, which is distant from the active site and has been postulated to play a regulatory role. The heme in CBS is unusual; it is six-coordinate, low spin, and contains cysteine and histidine as axial ligands. In this study, we have assessed the redox behavior of a human CBS dimeric variant lacking the C-terminal regulatory domain. Potentiometric redox titrations showed a reversible response with a reduction potential of -291 +/- 5 mV versus the normal hydrogen electrode, at pH 7.2. Stopped-flow kinetic determinations demonstrated that Fe(II)CBS reacted with dioxygen yielding Fe(III)CBS without detectable formation of an intermediate species. A linear dependence of the apparent rate constant of Fe(II)CBS decay on dioxygen concentration was observed and yielded a second-order rate constant of (1.11 +/- 0.07) x 10 (5) M (-1) s (-1) at pH 7.4 and 25 degrees C for the direct reaction of Fe(II)CBS with dioxygen. A similar reactivity was observed for full-length CBS. Heme oxidation led to superoxide radical generation, which was detected by the superoxide dismutase (SOD)-inhibitable oxidation of epinephrine. Our results show that CBS may represent a previously unrecognized source of cytosolic superoxide radical.     

Solvent-accessible cysteines in human cystathionine beta-synthase: crucial role of cysteine 431 in S-adenosyl-L-methionine binding

Cystathionine beta-synthase (CBS) is a tetrameric heme protein that catalyzes the PLP-dependent condensation of serine and homocysteine to cystathionine. CBS occupies a crucial regulatory position between the methionine cycle and transsulfuration. Human CBS contains 11 cysteine residues that are highly conserved in mammals but completely absent in the yeast enzyme, which catalyzes an identical reaction, suggesting a possible regulatory role for some of these residues. In this report, we demonstrate that in both the presence and absence of the CBS allosteric regulator S-adenosyl-l-methionine (AdoMet), only C15 and C431 of human CBS are solvent accessible. Mutagenesis of C15 to serine did not affect catalysis or AdoMet activation but significantly reduced aggregation of the purified enzyme in vitro. Mutagenesis of C431 resulted in a constitutively activated form of CBS that could not be further activated by either AdoMet or thermal activation. We and others have previously reported a number of C-terminal CBS point mutations that result in a decreased or abolished response to AdoMet. In contrast to all of these previously investigated CBS mutants, the C431 mutant form of CBS was unable to bind AdoMet, indicating that either this residue is directly involved in AdoMet binding or its absence induces a conformational change that destroys the integrity of the binding site for this regulatory ligand.     

Characterization of transsulfuration and cysteine biosynthetic pathways in the protozoan hemoflagellate, Trypanosoma cruzi. Isolation and molecular characterization of cystathionine beta-synthase and serine acetyltransferase from Trypanosoma

Sulfur-containing amino acids play an important role in a variety of cellular functions such as protein synthesis, methylation, and polyamine and glutathione synthesis. We cloned and characterized cDNA encoding cystathionine beta-synthase (CBS), which is a key enzyme of transsulfuration pathway, from a hemoflagellate protozoan parasite Trypanosoma cruzi. T. cruzi CBS, unlike mammalian CBS, lacks the regulatory carboxyl terminus, does not contain heme, and is not activated by S-adenosylmethionine. T. cruzi CBS mRNA is expressed as at least six independent isotypes with sequence microheterogeneity from tandemly linked multicopy genes. The enzyme forms a homotetramer and, in addition to CBS activity, the enzyme has serine sulfhydrylase and cysteine synthase (CS) activities in vitro. Expression of the T. cruzi CBS in Saccharomyces cerevisiae and Escherichia coli demonstrates that the CBS and CS activities are functional in vivo. Enzymatic studies on T. cruzi extracts indicate that there is an additional CS enzyme and stage-specific control of CBS and CS expression. We also cloned and characterized cDNA encoding serine acetyltransferase (SAT), a key enzyme in the sulfate assimilatory cysteine biosynthetic pathway. Dissimilar to bacterial and plant SAT, a recombinant T. cruzi SAT showed allosteric inhibition by l-cysteine, l-cystine, and, to a lesser extent, glutathione. Together, these studies demonstrate the T. cruzi is a unique protist in possessing both transsulfuration and sulfur assimilatory pathways.     

The redox behavior of the heme in cystathionine beta-synthase is sensitive to pH

Human cystathionine beta-synthase (CBS) is a unique pyridoxal-5'-phosphate-dependent enzyme in which heme is also present as a cofactor. Because the function of heme in this enzyme has yet to be elucidated, the study presented herein investigated possible relationships between the chemistry of the heme and the strong pH dependence of CBS activity. This study revealed, via study of a truncation variant, that the catalytic core of the enzyme governs the pH dependence of the activity. The heme moiety was found to play no discernible role in regulating CBS enzyme activity by sensing changes in pH, because the coordination sphere of the heme is not altered by changes in pH over a range of pH 6-9. Instead, pH was found to control the equilibrium amount of ferric and ferrous heme present after reaction of CBS with one-electron reducing agents. A variety of spectroscopic techniques, including resonance Raman, magnetic circular dichroism, and electron paramagnetic resonance, demonstrated that at pH 9 Fe(II) CBS is dominant while at pH 6 Fe(III) CBS is favored. At low pH, Fe(II) CBS forms transiently but reoxidizes by an apparent proton-gated electron-transfer mechanism. Regulation of CBS activity by the iron redox state has been proposed as the role of the heme moiety in this enzyme. Given that the redox behavior of the CBS heme appears to be controlled by pH, interplay of pH and oxidation state effects must occur if CBS activity is redox regulated.     

Surrogate genetics and metabolic profiling for characterization of human disease alleles

Cystathionine-β-synthase (CBS) deficiency is a human genetic disease causing homocystinuria, thrombosis, mental retardation, and a suite of other devastating manifestations. Early detection coupled with dietary modification greatly reduces pathology, but the response to treatment differs with the allele of CBS. A better understanding of the relationship between allelic variants and protein function will improve both diagnosis and treatment. To this end, we tested the function of 84 CBS alleles previously sequenced from patients with homocystinuria by ortholog replacement in Saccharomyces cerevisiae. Within this clinically associated set, 15% of variant alleles were indistinguishable from the predominant CBS allele in function, suggesting enzymatic activity was retained. An additional 37% of the alleles were partially functional or could be rescued by cofactor supplementation in the growth medium. This large class included alleles rescued by elevated levels of the cofactor vitamin B6, but also alleles rescued by elevated heme, a second CBS cofactor. Measurement of the metabolite levels in CBS-substituted yeast grown with different B6 levels using LC-MS revealed changes in metabolism that propagated beyond the substrate and product of CBS. Production of the critical antioxidant glutathione through the CBS pathway was greatly decreased when CBS function was restricted through genetic, cofactor, or substrate restriction, a metabolic consequence with implications for treatment.     

Pre-steady-state kinetic analysis of enzyme-monitored turnover during cystathionine β-synthase-catalyzed H(2)S generation

Cystathionine β-synthase (CBS) catalyzes the first step in the transsulfuration pathway in mammals, i.e., the condensation of serine and homocysteine to produce cystathionine and water. Recently, we have reported a steady-state kinetic analysis of the three hydrogen sulfide (H(2)S)-generating reactions that are catalyzed by human and yeast CBS [Singh, S., et al. (2009) J. Biol. Chem. 284, 22457-22466]. In the study presented here, we report a pre-steady-state kinetic analysis of intermediates in the H(2)S-generating reactions catalyzed by yeast CBS (yCBS). Because yCBS does not have a heme cofactor, in contrast to human CBS, it is easier to observe reaction intermediates with yCBS. The most efficient route for H(2)S generation by yCBS is the β-replacement of the cysteine thiol with homocysteine. In this reaction, yCBS first reacts with cysteine to release H(2)S and forms an aminoacrylate intermediate (k(obs) of 1.61 ± 0.04 mM(-1) s(-1) at low cysteine concentrations and 2.8 ± 0.1 mM(-1) s(-1) at high cysteine concentrations, at 20 °C), which has an absorption maximum at 465 nm. Homocysteine binds to the E·aminoacrylate intermediate with a bimolecular rate constant of 142 mM(-1) s(-1) and rapidly condenses to form the enzyme-bound external aldimine of cystathionine. The reactions could be partially rate limited by release of the products, cystathionine and H(2)S.     

Identification of rate-limiting steps in yeast heme biosynthesis

The heme biosynthesis pathway in the yeast Saccharomyces cerevisiae is a highly regulated system, but the mechanisms accounting for this regulation remain unknown. In an attempt to identify rate-limiting steps in heme synthesis, which may constitute potential regulatory points, we constructed yeast strains overproducing two enzymes of the pathway: the porphobilinogen synthase (PBG-S) and deaminase (PBG-D). Biochemical analysis of the enzyme-overproducing strains revealed intracellular porphobilinogen and porphyrin accumulation. These results indicate that both enzymes play a rate-limiting role in yeast heme biosynthesis.     

Vascular complications of cystathionine β-synthase deficiency: future directions for homocysteine-to-hydrogen sulfide research

Homocysteine (Hcy), a cardiovascular and neurovascular disease risk factor, is converted to hydrogen sulfide (H(2)S) through the transsulfuration pathway. H(2)S has attracted considerable attention in recent years for many positive effects on vascular health and homeostasis. Cystathionine β-synthase (CBS) is the first, and rate-limiting, enzyme in the transsulfuration pathway. Mutations in the CBS gene decrease enzymatic activity, which increases the plasma Hcy concentration, a condition called hyperhomocysteinemia (HHcy). Animal models of CBS deficiency have provided invaluable insights into the pathological effects of transsulfuration impairment and of both mild and severe HHcy. However, studies have also highlighted the complexity of HHcy and the need to explore the specific details of Hcy metabolism in addition to Hcy levels per se. There has been a relative paucity of work addressing the dysfunctional H(2)S production in CBS deficiency that may contribute to, or even create, HHcy-associated pathologies. Experiments using CBS knockout mice, both homozygous (-/-) and heterozygous (+/-), have provided 15 years of new knowledge and are the focus of this review. These murine models present the opportunity to study a specific mechanism for HHcy that matches one of the etiologies in many human patients. Therefore, the goal of this review was to integrate and highlight the critical information gained thus far from models of CBS deficiency and draw attention to critical gaps in knowledge, with particular emphasis on the modulation of H(2)S metabolism. We include findings from human and animal studies to identify important opportunities for future investigation that should be aimed at generating new basic and clinical understanding of the role of CBS and transsulfuration in cardiovascular and neurovascular disease.     

Deciphering the hybridisation history leading to the Lager lineage based on the mosaic genomes of Saccharomyces bayanus strains NBRC1948 and CBS380

Saccharomyces bayanus is a yeast species described as one of the two parents of the hybrid brewing yeast S. pastorianus. Strains CBS380(T) and NBRC1948 have been retained successively as pure-line representatives of S. bayanus. In the present study, sequence analyses confirmed and upgraded our previous finding: S. bayanus type strain CBS380(T) harbours a mosaic genome. The genome of strain NBRC1948 was also revealed to be mosaic. Both genomes were characterized by amplification and sequencing of different markers, including genes involved in maltotriose utilization or genes detected by array-CGH mapping. Sequence comparisons with public Saccharomyces spp. nucleotide sequences revealed that the CBS380(T) and NBRC1948 genomes are composed of: a predominant non-cerevisiae genetic background belonging to S. uvarum, a second unidentified species provisionally named S. lagerae, and several introgressed S. cerevisiae fragments. The largest cerevisiae-introgressed DNA common to both genomes totals 70kb in length and is distributed in three contigs, cA, cB and cC. These vary in terms of length and presence of MAL31 or MTY1 (maltotriose-transporter gene). In NBRC1948, two additional cerevisiae-contigs, cD and cE, totaling 12kb in length, as well as several smaller cerevisiae fragments were identified. All of these contigs were partially detected in the genomes of S. pastorianus lager strains CBS1503 (S. monacensis) and CBS1513 (S. carlsbergensis) explaining the noticeable common ability of S. bayanus and S. pastorianus to metabolize maltotriose. NBRC1948 was shown to be inter-fertile with S. uvarum CBS7001. The cross involving these two strains produced F1 segregants resembling the strains CBS380(T) or NRRLY-1551. This demonstrates that these S. bayanus strains were the offspring of a cross between S. uvarum and a strain similar to NBRC1948. Phylogenies established with selected cerevisiae and non-cerevisiae genes allowed us to decipher the complex hybridisation events linking S. lagerae/S. uvarum/S. cerevisiae with their hybrid species, S. bayanus/pastorianus.     

Effect of the Disease-Causing R266K Mutation on the Heme and PLP Environments of Human Cystathionine β-Synthase

Cystathionine β-synthase (CBS) is an essential pyridoxal 5'-phosphate (PLP)-dependent enzyme of the transsulfuration pathway that condenses serine with homocysteine to form cystathionine; intriguingly, human CBS also contains a heme b cofactor of unknown function. Herein we describe the enzymatic and spectroscopic properties of a disease-associated R266K hCBS variant, which has an altered hydrogen-bonding environment. The R266K hCBS contains a low-spin, six-coordinate Fe(III) heme bearing a His/Cys ligation motif, like that of WT hCBS; however, there is a geometric distortion that exists at the R266K heme. Using rR spectroscopy, we show that the Fe(III)-Cys(thiolate) bond is longer and weaker in R266K, as evidenced by an 8 cm(-1) downshift in the ν(Fe-S) resonance. Presence of this longer and weaker Fe(III)-Cys(thiolate) bond is correlated with alteration of the fluorescence spectrum of the active PLP ketoenamine tautomer. Activity data demonstrate that, relative to WT, the R266K variant is more impaired in the alternative cysteine-synthesis reaction than in the canonical cystathionine-synthesis reaction. This diminished cysteine synthesis activity and a greater sensitivity to exogenous PLP correlate with the change in PLP environment. Fe-S(Cys) bond weakening causes a nearly 300-fold increase in the rate of ligand switching upon reduction of the R266K heme. Combined, these data demonstrate cross talk between the heme and PLP active sites, consistent with previous proposals, revealing that alteration of the Arg(266)-Cys(52) interaction affects PLP-dependent activity and dramatically destabilizes the ferrous thiolate-ligated heme complex, underscoring the importance of this hydrogen-bonding residue pair.     

Ferrous human cystathionine beta-synthase loses activity during enzyme assay due to a ligand switch process

Cystathionine beta-synthase (CBS) is a pyridoxal-5'-phosphate-dependent enzyme that catalyzes the condensation of serine and homocysteine to form cystathionine. Mammalian CBS also contains a heme cofactor that has been proposed to allosterically regulate enzyme activity via the heme redox state, with FeII CBS displaying approximately half the activity of FeIII CBS in vitro. The results of this study show that human FeII CBS spontaneously loses enzyme activity over the course of a 20 min enzyme assay. Both the full-length 63-kDa and truncated 45-kDa form of CBS slowly and irreversibly lose activity upon reduction to the FeII form. Additionally, electronic absorption spectroscopy reveals that FeII CBS undergoes a heme ligand exchange to FeII CBS424 when the enzyme is incubated at 37 degrees C and pH 8.6. The addition of enzyme substrates or imidazole has a moderate effect on the rate of the ligand switch, but does not prevent conversion to the inactive species. Time-dependent spectroscopic data describing the conversion of FeII CBS to FeII CBS424 were fitted to a three-state kinetic model. The resultant rate constants were used to fit assay data and to estimate the activity of FeII CBS prior to the ligand switch. Based on this fit it appears that FeII CBS initially has the same enzyme activity as FeIII CBS, but FeII CBS loses activity as the ligand switch proceeds. The slow and irreversible loss of FeII CBS enzyme activity in vitro resembles protein denaturation, and suggests that a simple regulatory mechanism based on the heme redox state is unlikely.     

Allosteric Communication between the Pyridoxal 5′-Phosphate (PLP) and Heme Sites in the H2S Generator Human Cystathionine β-Synthase

Human cystathionine β-synthase (CBS) is a unique pyridoxal 5′-phosphate (PLP)-dependent enzyme that has a regulatory heme cofactor. Previous studies have demonstrated the importance of Arg-266, a residue at the heme pocket end of α-helix 8, for communication between the heme and PLP sites. In this study, we have examined the role of the conserved Thr-257 and Thr-260 residues, located at the other end of α-helix 8 on the heme electronic environment and on activity. The mutations at the two positions destabilize PLP binding, leading to lower PLP content and ∼2- to ∼500-fold lower activity compared with the wild-type enzyme. Activity is unresponsive to PLP supplementation, consistent with the pyridoxine-nonresponsive phenotype of the T257M mutation in a homocystinuric patient. The H₂S-producing activities, also impacted by the mutations, show a different pattern of inhibition compared with the canonical transsulfuration reaction. Interestingly, the mutants exhibit contrasting sensitivities to the allosteric effector, S-adenosylmethionine (AdoMet); whereas T257M and T257I are inhibited, the other mutants are hyperactivated by AdoMet. All mutants showed an increased propensity of the ferrous heme to form an inactive species with a 424 nm Soret peak and exhibited significantly reduced enzyme activity in the ferrous and ferrous-CO states. Our results provide the first evidence for bidirectional transmission of information between the cofactor binding sites, suggest the additional involvement of this region in allosteric communication with the regulatory AdoMet-binding domain, and reveal the potential for independent modulation of the canonical transsulfuration versus H₂S-generating reactions catalyzed by CBS.     

Domain Organization,Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases

Cystathionine beta-synthase (CBS) is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS), fruit fly (dCBS) and yeast (yCBS) enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme’s catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet); however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme’s catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.     

Yeast cystathionine beta-synthase is a pyridoxal phosphate enzyme but, unlike the human enzyme, is not a heme protein

Our studies of cystathionine beta-synthase from Saccharomyces cerevisiae (yeast) are aimed at clarifying the cofactor dependence and catalytic mechanism and obtaining a system for future investigations of the effects of mutations that cause human disease (homocystinuria or coronary heart disease). We report methods that yielded high expression of the yeast gene in Escherichia coli and of purified yeast cystathionine beta-synthase. The absorption and circular dichroism spectra of the homogeneous enzyme were characteristic of a pyridoxal phosphate enzyme and showed the absence of heme, which is found in human and rat cystathionine beta-synthase. The absence of heme in the yeast enzyme facilitates spectroscopic studies to probe the catalytic mechanism. The reaction of the enzyme with L-serine in the absence of L-homocysteine produced the aldimine of aminoacrylate, which absorbed at 460 nm and had a strong negative circular dichroism band at 460 nm. The formation of this intermediate from the product, L-cystathionine, demonstrates the partial reversibility of the reaction. Our results establish the overall catalytic mechanism of yeast cystathionine beta-synthase and provide a useful system for future studies of structure and function. The absence of heme in the functional yeast enzyme suggests that heme does not play an essential catalytic role in the rat and human enzymes. The results are consistent with the absence of heme in the closely related enzymes O-acetylserine sulfhydrylase, threonine deaminase, and tryptophan synthase.     

Combination of 10% EDTA, Photosan, and a blue light hand-held photopolymerizer to inactivate leading oral bacteria in dentistry in vitro

Aims:  To study the yeast diversity of Nigerian palm wines by comparison with other African strains.
Methods and Results:  Twenty-three Saccharomyces cerevisiae strains were obtained from palm wine samples collected at four locations in eastern Nigeria, and characterized using different molecular techniques: internal transcribed spacer restriction fragment length polymorphism and sequence analysis, pulsed field gel electrophoresis, inter delta typing and microsatellite multilocus analysis. These techniques revealed that palm wine yeasts represent a group of closely related strains that includes other West African isolates (CBS400, NCYC110, DVPG6044). Population analysis revealed an excess of homozygote strains and an allelic richness similar to wine suggestive of local domestication. Several other African yeast strains were not connected to this group. Ghana sorghum beer strains and other African strains (DBVPG1853 and MUCL28071) displayed strikingly high relatedness with European bread, beer or wine strains, and the genome of strain MUCL30909 contained African and wine-type alleles, indicating its hybrid origin.
Conclusions:  Nigerian palm wine yeast represents a local specific yeast flora, whereas a European origin or hybrid was suspected for several other Africa isolates.
Significance and Impact of the Study:  This study presents the first genetic characterization of an autochthonous African palm wine yeast population and confirms the idea that human intervention has favoured yeast migration.