A diagnostic test that uses isothermal amplification and lateral flow detection sdaA can detect tuberculosis in 60 min.

AIMS: Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is now the leading cause of death from infectious disease, thus rapid diagnostic and screening techniques for TB are urgently needed. METHODS AND RESULTS: Here, a detection of MTB using multiple cross displacement amplification coupling with nanoparticles-based lateral flow device (MCDA-LFD) was developed and validated, targeting the specific sdaA gene. The whole detection procedure, including rapid genomic DNA extraction (15 min), amplification (30 min) and result reporting (2 min), was completed within 50 min. No cross-reaction with non-mycobacteria and non-tuberculous mycobacteria (NTM) strains was observed. The sensitivity of sdaA-MCDA-LFD, Xpert MTB/RIF assay and culture results was 81·6, 48·3 and 37·9%, respectively, in TB patients. Among positive culture samples, the sensitivity of sdaA-MCDA-LFD and Xpert MTB/RIF assay was 93·9% (31/33) and 81·8% (27/33), respectively. Among culture-negative samples, the sensitivity of sdaA-MCDA-LFD and Xpert MTB/RIF assay was 74·1% (40/54) and 27·8% (15/54), respectively. The specificity of sdaA-MCDA-LFD and Xpert MTB/RIF was 95·4% (62/65) and 100% (65/65) in clinical samples from non-TB patients. CONCLUSION: The sdaA-MCDA-LFD assay was a rapid, simple, specific and sensitive TB diagnostic test. SIGNIFICANCE AND IMPACT OF THE STUDY: The sdaA-MCDA-LFD assay holds promise for application as a useful point-of-care test to detect MTB, and will play an important role in controlling and preventing TB.

Serine metabolism in the brain regulates starvation-induced sleep suppression in Drosophila melanogaster.

Sleep and metabolism are physiologically and behaviorally intertwined; however, the molecular basis for their interaction remains poorly understood. Here, we identified a serine metabolic pathway as a key mediator for starvation-induced sleep suppression. Transcriptome analyses revealed that enzymes involved in serine biosynthesis were induced upon starvation in Drosophila melanogaster brains. Genetic mutants of astray (aay), a fly homolog of the rate-limiting phosphoserine phosphatase in serine biosynthesis, displayed reduced starvation-induced sleep suppression. In contrast, a hypomorphic mutation in a serine/threonine-metabolizing enzyme, serine/threonine dehydratase (stdh), exaggerated starvation-induced sleep suppression. Analyses of double mutants indicated that aay and stdh act on the same genetic pathway to titrate serine levels in the head as well as to adjust starvation-induced sleep behaviors. RNA interference-mediated depletion of aay expression in neurons, using cholinergic Gal4 drivers, phenocopied aay mutants, while a nicotinic acetylcholine receptor antagonist selectively rescued the exaggerated starvation-induced sleep suppression in stdh mutants. Taken together, these data demonstrate that neural serine metabolism controls sleep during starvation, possibly via cholinergic signaling. We propose that animals have evolved a sleep-regulatory mechanism that reprograms amino acid metabolism for adaptive sleep behaviors in response to metabolic needs.

Analyses of variants of the Ser/Thr dehydratase IlvA provide insight into 2-aminoacrylate metabolism in Salmonella enterica.

RidA is a conserved and broadly distributed protein that has enamine deaminase activity. In a variety of organisms tested thus far, lack of RidA results in the accumulation of the reactive metabolite 2-aminoacrylate (2AA), an obligate intermediate in the catalytic mechanism of several pyridoxal 5'-phosphate (PLP)-dependent enzymes. This study reports the characterization of variants of the biosynthetic serine/threonine dehydratase (EC 4.3.1.19; IlvA), which is a significant generator of 2AA in the bacteria Salmonella enterica, Escherichia coli, and Pseudomonas aeruginosa and the yeast Saccharomyces cerevisiae Two previously identified mutations, ilvA3210 and ilvA3211, suppressed the phenotypic growth consequences of 2AA accumulation in S. enterica Characterization of the respective protein variants suggested that they affect 2AA metabolism in vivo by two different catalytic mechanisms, both leading to an overall reduction in serine dehydratase activity. To emphasize the physiological relevance of the in vitro enzyme characterization, we sought to explain in vivo phenotypes using these data. A simple mathematical model describing the impact these catalytic deficiencies had on 2AA production was generally supported by our data. However, caveats arose when kinetic parameters, determined in vitro, were used to predict formation of the isoleucine precursor 2-ketobutyrate and model in vivo (growth) behaviors. Altogether, our data support the need for a holistic approach, including in vivo and in vitro analyses, to generate data used in understanding and modeling metabolism.

Structural and biochemical studies on the role of active site Thr166 and Asp236 in the catalytic function of D-Serine deaminase from Salmonella typhimurium.

D-Serine deaminase (DSD) degrades D-Ser to pyruvate and ammonia. Uropathogenic bacteria survive in the toxic D-Ser containing mammalian urine because of DSD activity. The crystal structure of the apo form of Salmonella typhimurium DSD (StDSD) has been reported earlier. In the present work, we have investigated the role of two active site residues, Thr166 and Asp236 by site directed mutagenesis (T166A and D236L). The enzyme activity is lost upon mutation of these residues. The 2.7 Šresolution crystal structure of T166A DSD with bound PLP reported here represents the first structure of the holo form of StDSD. PLP binding induces small changes in the relative dispositions of the minor and major domains of the protein and this inter-domain movement becomes substantial upon interaction with the substrate. The conformational changes bring Thr166 to a position at the active site favorable for the degradation of D-Ser. Examination of the different forms of the enzyme and comparison with structures of homologous enzymes suggests that Thr166 is the most probable base abstracting proton from the Cα atom of the substrate and Asp236 is crucial for binding of the cofactor.

The Response to 2-Aminoacrylate Differs in Escherichia coli and Salmonella enterica, despite Shared Metabolic Components.

The metabolic network of an organism includes the sum total of the biochemical reactions present. In microbes, this network has an impeccable ability to sense and respond to perturbations caused by internal or external stimuli. The metabolic potential (i.e., network structure) of an organism is often drawn from the genome sequence, based on the presence of enzymes deemed to indicate specific pathways. Escherichia coli and Salmonella enterica are members of the Enterobacteriaceae family of Gram-negative bacteria that share the majority of their metabolic components and regulatory machinery as the "core genome." In S. enterica, the ability of the enamine intermediate 2-aminoacrylate (2AA) to inactivate a number of pyridoxal 5'-phosphate (PLP)-dependent enzymes has been established in vivo In this study, 2AA metabolism and the consequences of its accumulation were investigated in E. coli The data showed that despite the conservation of all relevant enzymes, S. enterica and E. coli differed in both the generation and detrimental consequences of 2AA. In total, these findings suggest that the structure of the metabolic network surrounding the generation and response to endogenous 2AA stress differs between S. enterica and E. coliIMPORTANCE This work compared the metabolic networks surrounding the endogenous stressor 2-aminoacrylate in two closely related members of the Enterobacteriaceae The data showed that despite the conservation of all relevant enzymes in this metabolic node, the two closely related organisms diverged in their metabolic network structures. This work highlights how a set of conserved components can generate distinct network architectures and how this can impact the physiology of an organism. This work defines a model to expand our understanding of the 2-aminoacrylate stress response and the differences in metabolic structures and cellular milieus between S. enterica and E. coli.

Arabidopsis and maize RidA proteins preempt reactive enamine/imine damage to branched-chain amino acid biosynthesis in plastids.

RidA (for Reactive Intermediate Deaminase A) proteins are ubiquitous, yet their function in eukaryotes is unclear. It is known that deleting Salmonella enterica ridA causes Ser sensitivity and that S. enterica RidA and its homologs from other organisms hydrolyze the enamine/imine intermediates that Thr dehydratase forms from Ser or Thr. In S. enterica, the Ser-derived enamine/imine inactivates a branched-chain aminotransferase; RidA prevents this damage. Arabidopsis thaliana and maize (Zea mays) have a RidA homolog that is predicted to be plastidial. Expression of either homolog complemented the Ser sensitivity of the S. enterica ridA mutant. The purified proteins hydrolyzed the enamines/imines formed by Thr dehydratase from Ser or Thr and protected the Arabidopsis plastidial branched-chain aminotransferase BCAT3 from inactivation by the Ser-derived enamine/imine. In vitro chloroplast import assays and in vivo localization of green fluorescent protein fusions showed that Arabidopsis RidA and Thr dehydratase are chloroplast targeted. Disrupting Arabidopsis RidA reduced root growth and raised the root and shoot levels of the branched-chain amino acid biosynthesis intermediate 2-oxobutanoate; Ser treatment exacerbated these effects in roots. Supplying Ile reversed the root growth defect. These results indicate that plastidial RidA proteins can preempt damage to BCAT3 and Ile biosynthesis by hydrolyzing the Ser-derived enamine/imine product of Thr dehydratase.

Mutagenic and chemical analyses provide new insight into enzyme activation and mechanism of the type 2 iron-sulfur l-serine dehydratase from Legionella pneumophila.

The crystal structure of the Type 2 l-serine dehydratase from Legionella pneumophila (lpLSD), revealed a "tail-in-mouth" configuration where the C-terminal residue acts as an intrinsic competitive inhibitor. This pre-catalytic structure undergoes an activation step prior to catalytic turnover. Mutagenic analysis of residues at or near the active site cleft is consistent with stabilization of substrate binding by many of the same residues that interact with the C-terminal cysteine and highlight the critical role of certain tail residues in activity. pH-rate profiles show that a residue with pK of 5.9 must be deprotonated and a residue with a pK of 8.5 must be protonated for activity. This supports an earlier suggestion that His 61 is the likely catalytic base. An additional residue with a pK of 8.5-9 increases cooperativity when it is deprotonated. This investigation also demonstrates that the Fe-S dehydratases convert the enamine/imine intermediates of the catalytic reaction to products on the enzyme prior to release. This is in contrast to pyridoxyl 5' phosphate based dehydratases that release an enamine/imine intermediate into solution, which then hydrolyzes to produce the ketoamine product.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Unique Conformational and Chemical Transformations Occurring upon [4Fe-4S] Cluster Binding in the Type 2 L-Serine Dehydratase from Legionella pneumophila.

The type 2 L-serine dehydratase from Legionella pneumophila (lpLSD) contains a [4Fe-4S](2+) cluster that acts as a Lewis acid to extract the hydroxyl group of L-serine during the dehydration reaction. Surprisingly, the crystal structure shows that all four of the iron atoms in the cluster are coordinated with protein cysteinyl residues and that the cluster is buried and not exposed to solvent. If the crystal structure of lpLSD accurately reflects the structure in solution, then substantial rearrangement at the active site is necessary for the substrate to enter. Furthermore, repair of the oxidized protein when the cluster has degraded would presumably entail exposure of the buried cysteine ligands. Thus, the conformation required for the substrate to enter may be similar to those required for a new cluster to enter the active site. To address this, hydrogen-deuterium exchange combined with mass spectrometry (HDX MS) was used to probe the conformational changes that occur upon oxidative degradation of the Fe-S cluster. The regions that show the most significant differential HDX are adjacent to the cluster location in the holoenzyme or connect regions that are adjacent to the cluster. The observed decrease in flexibility upon cluster binding provides direct evidence that the "tail-in-mouth" conformation observed in the crystal structure also occurs in solution and that the C-terminal peptide is coordinated to the [4Fe-4S] cluster in a precatalytic conformation. This observation is consistent with the requirement of an activation step prior to catalysis and the unusually high level of resistance to oxygen-induced cluster degradation. Furthermore, peptide mapping of the apo form under nonreducing conditions revealed the formation of disulfide bonds between C396 and C485 and possibly between C343 and C385. These observations provide a picture of how the cluster loci are stabilized and poised to receive the cluster in the apo form and the requirement for a reduction step during cluster formation.

Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei.

L-serine ammonia-lyase, as a member of the ß-family of pyridoxal-5'-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic ß-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/ß structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0-9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of ß-elimination enzymes and will provide a basis for further enzyme engineering.

Structure of L-serine dehydratase from Legionella pneumophila: novel use of the C-terminal cysteine as an intrinsic competitive inhibitor.

Here we report the first complete structure of a bacterial Fe-S l-serine dehydratase determined to 2.25 Å resolution. The structure is of the type 2 l-serine dehydratase from Legionella pneumophila that consists of a single polypeptide chain containing a catalytic α domain and a ß domain that is structurally homologous to the "allosteric substrate binding" or ASB domain of d-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis. The enzyme exists as a dimer of identical subunits, with each subunit exhibiting a bilobal architecture. The [4Fe-4S](2+) cluster is bound by residues from the C-terminal α domain and is situated between this domain and the N-terminal ß domain. Remarkably, the model reveals that the C-terminal cysteine residue (Cys 458), which is conserved among the type 2 l-serine dehydratases, functions as a fourth ligand to the iron-sulfur cluster producing a "tail in mouth" configuration. The interaction of the sulfhydryl group of Cys 458 with the fourth iron of the cluster appears to mimic the position that the substrate would adopt prior to catalysis. A number of highly conserved or invariant residues found in the ß domain are clustered around the iron-sulfur center. Ser 16, Ser 17, Ser 18, and Thr 290 form hydrogen bonds with the carboxylate group of Cys 458 and the carbonyl oxygen of Glu 457, whereas His 19 and His 61 are poised to potentially act as the catalytic base required for proton extraction. Mutation of His 61 produces an inactive enzyme, whereas the H19A protein variant retains substantial activity, suggesting that His 61 serves as the catalytic base. His 124 and Asn 126, found in an HXN sequence, point toward the Fe-S cluster. Mutational studies are consistent with these residues either binding a serine molecule that serves as an activator or functioning as a potential trap for Cys 458 as it moves out of the active site prior to catalysis.

Engineering of Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex to improve poly(3-hydroxybutyrate) production in Escherichia coli.

BACKGROUND: Poly(3-hydroxybutyrate) (PHB), a biodegradable bio-plastic, is one of the most common homopolymer of polyhydroxyalkanoates (PHAs). PHB is synthesized by a variety of microorganisms as intracellular carbon and energy storage compounds in response to environmental stresses. Bio-based production of PHB from renewable feedstock is a promising and sustainable alternative to the petroleum-based chemical synthesis of plastics. In this study, a novel strategy was applied to improve the PHB biosynthesis from different carbon sources. RESULTS: In this research, we have constructed E. coli strains to produce PHB by engineering the Serine-Deamination (SD) pathway, the Entner-Doudoroff (ED) pathway, and the pyruvate dehydrogenase (PDH) complex. Firstly, co-overexpression of sdaA (encodes L-serine deaminase), L-serine biosynthesis genes and pgk (encodes phosphoglycerate kinase) activated the SD Pathway, and the resulting strain SD02 (pBHR68), harboring the PHB biosynthesis genes from Ralstonia eutropha, produced 4.86 g/L PHB using glucose as the sole carbon source, representing a 2.34-fold increase compared to the reference strain. In addition, activating the ED pathway together with overexpressing the PDH complex further increased the PHB production to 5.54 g/L with content of 81.1% CDW. The intracellular acetyl-CoA concentration and the [NADPH]/[NADP(+)] ratio were enhanced after the modification of SD pathway, ED pathway and the PDH complex. Meanwhile, these engineering strains also had a significant increase in PHB concentration and content when xylose or glycerol was used as carbon source. CONCLUSIONS: Significant levels of PHB biosynthesis from different kinds of carbon sources can be achieved by engineering the Serine-Deamination pathway, Entner-Doudoroff pathway and pyruvate dehydrogenase complex in E. coli JM109 harboring the PHB biosynthesis genes from Ralstonia eutropha. This work demonstrates a novel strategy for improving PHB production in E. coli. The strategy reported here should be useful for the bio-based production of PHB from renewable resources.

Sphingoid bases and the serine catabolic enzyme CHA1 define a novel feedforward/feedback mechanism in the response to serine availability.

Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.

Catabolism of serine by Pediococcus acidilactici and Pediococcus pentosaceus.

The ability to produce diacetyl from pyruvate and l-serine was studied in various strains of Pediococcus pentosaceus and Pediococcus acidilactici isolated from cheese. After being incubated on both substrates, only P. pentosaceus produced significant amounts of diacetyl. This property correlated with measurable serine dehydratase activity in cell extracts. A gene encoding the serine dehydratase (dsdA) was identified in P. pentosaceus, and strains that showed no serine dehydratase activity carried mutations that rendered the gene product inactive. A functional dsdA was cloned from P. pentosaceus FAM19132 and expressed in Escherichia coli. The purified recombinant enzyme catalyzed the formation of pyruvate from L- and D-serine and was active at low pH and elevated NaCl concentrations, environmental conditions usually present in cheese. Analysis of the amino acid profiles of culture supernatants from dsdA wild-type and dsdA mutant strains of P. pentosaceus did not show differences in serine levels. In contrast, P. acidilactici degraded serine. Moreover, this species also catabolized threonine and produced alanine and α-aminobutyrate.

Identification and characterization of two new types of bacterial L-serine dehydratases and assessment of the function of the ACT domain.

Two new types of bacterial Fe-S L-serine dehydratases have been identified. These join two previously recognized enzyme types, for a total of four, that are distinguished on the basis of domain arrangement and amino acid sequence. A Type 3 enzyme from Amphibacillus xylanus (axLSD) and a Type 4 enzyme from Heliscomenobacter hydrossis (hhLSD) were cloned, expressed, purified, and characterized. Like the Type 1 enzyme from Bacillus subtilis (bsLSD), axLSD required a monovalent cation, preferably potassium, for activity. However, the hhLSD was without activity even after reconstitution of the iron-sulfur center by a process that successfully restored activity to oxygen-inactivated axLSD. This and other characteristics suggest that this Type 4 protein may be a pseudoenzyme. The oxygen sensitivity of axLSD was greater than other L-serine dehydratases so far studied and suggested that there may be significant conformational differences among the four types resulting in widely different solvent accessibility of the Fe-S clusters in these enzymes. The role of the ACT domain in these enzymes was explored by deleting it from bsLSD. Although there was an effect on the kinetic parameters, this domain was not responsible for the cation requirement nor did its removal have a significant effect on oxygen sensitivity.

The serine transporter SdaC prevents cell lysis upon glucose depletion in Escherichia coli.

The amino acid serine plays diverse metabolic roles, yet bacteria actively degrade exogenously provided serine via deamination to pyruvate. Serine deamination is thought to be a detoxification mechanism due to the ability of serine to inhibit several biosynthetic reactions, but this pathway remains highly active even in nutrient-replete conditions. While investigating the physiological roles of serine deamination in different growth conditions, we discovered that Escherichia coli cells lacking the sdaCB operon, which encodes the serine transporter SdaC and the serine deaminase SdaB, lyse upon glucose depletion in a medium containing no exogenous serine but all other amino acids and nucleobases. Unexpectedly, this lysis phenotype can be recapitulated by deleting sdaC alone and can be rescued by heterologous expression of SdaC. Lysis of ΔsdaC cells can be prevented by omitting glycine from the medium, inhibiting the glycine cleavage system, or by increasing alanine availability. Together, our results reveal that the serine transporter SdaC plays a critical role in maintaining amino acid homeostasis during shifts in nutrient availability in E. coli.

A catalytic mechanism that explains a low catalytic activity of serine dehydratase like-1 from human cancer cells: crystal structure and site-directed mutagenesis studies.

SDH (l-serine dehydratase, EC 4.3.1.17) is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that catalyzes dehydration of l-Ser/Thr to yield pyruvate/ketobutyrate and ammonia. A SDH isoform (cSDH) found in human cancer cell lines has relatively low catalytic activity in comparison with the liver enzyme (hSDH). The crystal structure of cSDH has been determined at 2.8 angstroms resolution. A PLP is covalently attached to K48 by Schiff-base linkage in the active site. The ring nitrogen of PLP is involved in a H-bonding with C309, but is apparently not protonated. Twenty-three amino residues that compose the active site surfaces were identified. The human and rat liver enzymes (hSDH and rSDH) have the same residues, while residues G72, A172, and S228 in cSDH are replaced with A66, S166, and A222, respectively, in hSDH. These residues in hSDH and cSDH were mutated to make complementary pairs of mutated enzymes, and their kinetic parameters were determined. C303 of hSDH and C309 of cSDH which are H-bonding partner of the ring nitrogen of PLP were mutated to alanine and their kinetic parameters were also determined. The crystal structures and the mutation data suggest that having a glycine at residue 72 of cSDH is the major reason for the reduction of catalytic activity of cSDH. Changing alanine to glycine at residue 72 increases the flexibility of the substrate binding-loop (71S(G/A)GN74), so that the bound substrate and PLP are not pushed deep into the active cleft. Consequently, the proton transfer rate from S(G) of C309 to N1 of the bound PLP is decreased, which determines the rate of catalytic reaction.

Deficiency in l-serine deaminase results in abnormal growth and cell division of Escherichia coli K-12.

The loss of the ability to deaminate l-serine severely impairs growth and cell division in Escherichia coli K-12. A strain from which the three genes (sdaA, sdaB, tdcG) coding for this organism's three l-serine deaminases had been deleted grows well in glucose minimal medium but, on subculture into minimal medium with glucose and casamino acids, it makes very large, abnormally shaped cells, many of which lyse. When inoculated into Luria-Bertani (LB) broth with or without glucose, it makes very long filaments. Provision of S-adenosylmethionine restores cell division in LB broth with glucose, and repairs much of the difficulty in growth in medium with casamino acids. We suggest that replication of E. coli is regulated by methylation, that an unusually high intracellular l-serine concentration, in the presence of other amino acids, starves the cell for S-adenosylmethionine and that it is the absence of S-adenosylmethionine and/or of C1-tetrahydrofolate derivatives that prevents normal cell division.

Dispersed mutations in histone H3 that affect transcriptional repression and chromatin structure of the CHA1 promoter in Saccharomyces cerevisiae.

The histone H3 amino terminus, but not that of H4, is required to prevent the constitutively bound activator Cha4 from remodeling chromatin and activating transcription at the CHA1 gene in Saccharomyces cerevisiae. Here we show that neither the modifiable lysine residues nor any specific region of the H3 tail is required for repression of CHA1. We then screened for histone H3 mutations that cause derepression of the uninduced CHA1 promoter and identified six mutants, three of which are also temperature-sensitive mutants and four of which exhibit a sin(-) phenotype. Histone mutant levels were similar to that of wild-type H3, and the mutations did not cause gross alterations in nucleosome structure. One specific and strongly derepressing mutation, H3 A111G, was examined in depth and found to cause a constitutively active chromatin configuration at the uninduced CHA1 promoter as well as at the ADH2 promoter. Transcriptional derepression and altered chromatin structure of the CHA1 promoter depend on the activator Cha4. These results indicate that modest perturbations in distinct regions of the nucleosome can substantially affect the repressive function of chromatin, allowing activation in the absence of a normal inducing signal (at CHA1) or of Swi/Snf (resulting in a sin(-) phenotype).

Enzymatic and biochemical properties of a novel human serine dehydratase isoform.

A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l-serine and l-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5'-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.

A regulatory element in the CHA1 promoter which confers inducibility by serine and threonine on Saccharomyces cerevisiae genes.

CHA1 of Saccharomyces cerevisiae is the gene for the catabolic L-serine (L-threonine) dehydratase, which is responsible for biodegradation of serine and threonine. We have previously shown that expression of the CHA1 gene is transcriptionally induced by serine and threonine. Northern (RNA) analysis showed that the additional presence of good nitrogen sources affects induction. This may well be due to inducer exclusion. To identify interactions of cis-acting elements with trans activators of the CHA1 promoter, we performed band shift assays of nuclear protein extracts with CHA1 promoter fragments. By this approach, we identified a protein-binding site of the CHA1 promoter. The footprint of this protein contains the ABF1-binding site consensus sequence. This in vitro binding activity is present irrespectively of CHA1 induction. By deletion analysis, two other elements of the CHA1 promoter, UAS1CHA and UAS2CHA, which are needed for induction of the CHA1 gene were identified. Each of the two sequence elements is sufficient to confer serine and threonine induction upon the CYC1 promoter when substituting its upstream activating sequence. Further, in a cha4 mutant strain which is unable to grow with serine or threonine as the sole nitrogen source, the function of UAS1CHA, as well as that of UAS2CHA, is obstructed.