Serine biosynthesis as a novel therapeutic target for dilated cardiomyopathy.

AIMS: Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro. METHODS AND RESULTS: Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions. CONCLUSIONS: A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.

The Serine Biosynthesis of Paenibacillus polymyxa WLY78 Is Regulated by the T-Box Riboswitch.

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.

Intracellular Trapping of the Selective Phosphoglycerate Dehydrogenase (PHGDH) Inhibitor BI-4924 Disrupts Serine Biosynthesis.

Phosphoglycerate dehydrogenase (PHGDH) is known to be the rate-limiting enzyme in the serine synthesis pathway in humans. It converts glycolysis-derived 3-phosphoglycerate to 3-phosphopyruvate in a co-factor-dependent oxidation reaction. Herein, we report the discovery of BI-4916, a prodrug of the co-factor nicotinamide adenine dinucleotide (NADH/NAD+)-competitive PHGDH inhibitor BI-4924, which has shown high selectivity against the majority of other dehydrogenase targets. Starting with a fragment-based screening, a subsequent hit optimization using structure-based drug design was conducted to deliver a single-digit nanomolar lead series and to improve potency by 6 orders of magnitude. To this end, an intracellular ester cleavage mechanism of the ester prodrug was utilized to achieve intracellular enrichment of the actual carboxylic acid based drug and thus overcome high cytosolic levels of the competitive cofactors NADH/NAD+.

Why is vitamin B6 effective in alleviating the symptoms of autism?

Many factors are reported to be involved in the complex pathophysiological processes of autism, suggesting that there is considerable variability in the manifestations of this disease. Several interventions are used to treat this disorder. Among them, vitamin B6 is widely used to treat the symptoms observed in autism. Vitamin B6 is beneficial for about half of autistic individuals in decreasing behavioral problems. However, until now, it remains unknown why vitamin B6 is effective for this disease. Although the exact pathogenesis is not defined, it is evident that certain neurotransmitter systems are impaired in the brains of autistic patients, causing the symptoms observed in the disease. In fact, impairment of many neurotransmitter systems has been reported, including GABA, serotonin, dopamine, and noradrenalin. Furthermore, vitamin B6 is important for the synthesis of many neurotransmitters, including GABA, serotonin, dopamine, noradrenalin, histamine, glycine, and d-serine, indicating that vitamin B6 supplementation may enhance many neurotransmitter systems. Thus, vitamin B6 supplementation can treat the impaired neurotransmitter systems in a given patient, even if the actual impaired neurotransmitter systems are not defined in that patient.

Disruption of De Novo Serine Synthesis in Müller Cells Induced Mitochondrial Dysfunction and Aggravated Oxidative Damage.

De novo serine synthesis plays important roles in normal mitochondrial function and cellular anti-oxidative capacity. It is reported to be mainly activated in glial cells of the central nervous system, but its role in retinal Müller glia remains unclear. In this study, we inhibited de novo serine synthesis using CBR-5884, a specific inhibitor of phosphoglycerate dehydrogenase (PHGDH, a rate limiting enzyme in de novo serine metabolism) in MIO-M1 cells (immortalized human Müller cells) and huPMCs (human primary Müller cells) under mild oxidative stress. Alamar blue and LDH (lactate dehydrogenase) assays showed significantly reduced metabolic activities and increased cellular damage of Müller cells, when exposed to CBR-5884 accompanied by mild oxidative stress; however, CBR-5884 alone had little effect. The increased cellular damage was partially reversed by supplementation with exogenous serine/glycine. HSP72 (an oxidative stress marker) and reactive oxygen species (ROS) levels were significantly increased; glutathione and NADPH/NADP+ levels were pronouncedly reduced under PHGDH inhibition accompanied by oxidative stress. JC-1 staining and Seahorse respiration experiments showed that inhibition of de novo serine synthesis in Müller cells can also increase mitochondrial stress and decrease mitochondrial ATP production. qPCR and Western blot demonstrated an increased expression of HSP60 (a key mitochondrial stress-related gene), and this was further validated in human retinal explants. Our study suggests that de novo serine synthesis is important for Müller cell survival, particularly when they are exposed to mild oxidative stress, possibly by maintaining mitochondrial function and generating glutathione and NADPH to counteract ROS.

Disturbed phospholipid metabolism in serine biosynthesis defects revealed by metabolomic profiling.

Serine biosynthesis defects are autosomal recessive metabolic disorders resulting from the deficiency of any of the three enzymes involved in de novo serine biosynthesis, specifically phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). In this study, we performed metabolomic profiling on 4 children with serine biosynthesis defects; 3 with PGDH deficiency and 1 with PSAT deficiency. The evaluations were performed at baseline and with serine and glycine supplementation. Metabolomic profiling performed at baseline showed low phospholipid species, including glycerophosphocholine, glycerophosphoethanolamine, and sphingomyelin. All children had low serine and glycine as expected. Low glycerophosphocholine compounds were found in 4 children, low glycerophosphoethanolamine compounds in 3 children, and low sphingomyelin species in 2 children. Metabolic profiling with serine and glycine supplementation showed normalization of most of the low phospholipid compounds in the 4 children. Phospholipids are the major component of plasma and intracellular membranes, and phosphatidylcholine is the most abundant phospholipid of all mammalian cell types and subcellular organelles. Phosphatidylcholine is of particular importance for the nervous system, where it is essential for neuronal differentiation. The observed low phosphatidylcholine species in children with serine biosynthesis defects that improved after serine supplementation, supports the role of serine as a significant precursor for phosphatidylcholine. The vital role that phosphatidylcholine has during neuronal differentiation and the pronounced neurological manifestations in serine biosynthesis defects suggest that phosphatidylcholine deficiency occurring secondary to serine deficiency may have a significant contribution to the development of the neurological manifestations in individuals with serine biosynthesis defects.

Studying the Function of the Phosphorylated Pathway of Serine Biosynthesis in Arabidopsis thaliana.

Photorespiration is an essential pathway in photosynthetic organisms and is particularly important to detoxify and recycle 2-phosphoglycolate (2-PG), a by-product of oxygenic photosynthesis. The enzymes that catalyze the reactions in the photorespiratory core cycle and closely associated pathways have been identified; however, open questions remain concerning the metabolic network in which photorespiration is embedded. The amino acid serine represents one of the major intermediates in the photorespiratory pathway and photorespiration is thought to be the major source of serine in plants. The restriction of photorespiration to autotrophic cells raises questions concerning the source of serine in heterotrophic tissues. Recently, the phosphorylated pathway of serine biosynthesis has been found to be extremely important for plant development and metabolism. In this protocol, we describe a detailed methodological workflow to analyze the generative and vegetative phenotypes of plants deficient in the phosphorylated pathway of serine biosynthesis, which together allow a better understanding of its function in plants.

The phosphorylated pathway of serine biosynthesis is essential both for male gametophyte and embryo development and for root growth in Arabidopsis.

This study characterizes the phosphorylated pathway of Ser biosynthesis (PPSB) in Arabidopsis thaliana by targeting phosphoserine phosphatase (PSP1), the last enzyme of the pathway. Lack of PSP1 activity delayed embryo development, leading to aborted embryos that could be classified as early curled cotyledons. The embryo-lethal phenotype of psp1 mutants could be complemented with PSP1 cDNA under the control of Pro35S (Pro35S:PSP1). However, this construct, which was poorly expressed in the anther tapetum, did not complement mutant fertility. Microspore development in psp1.1/psp1.1 Pro35S:PSP1 arrested at the polarized stage. The tapetum from these lines displayed delayed and irregular development. The expression of PSP1 in the tapetum at critical stages of microspore development suggests that PSP1 activity in this cell layer is essential in pollen development. In addition to embryo death and male sterility, conditional psp1 mutants displayed a short-root phenotype, which was reverted in the presence of Ser. A metabolomic study demonstrated that the PPSB plays a crucial role in plant metabolism by affecting glycolysis, the tricarboxylic acid cycle, and the biosynthesis of amino acids. We provide evidence of the crucial role of the PPSB in embryo, pollen, and root development and suggest that this pathway is an important link connecting primary metabolism with development.

[Metabolic flux analysis of L-serine synthesis by Corynebacterium glutamicum SYPS-062].

Corynebacterium glutamicum SYPS-062 was an L-serine producing strain stored at our lab and could produce L-serine directly from sugar. We studied the effects of cofactors in one carbon unit metabolism-folate and VB12 on the cell growth, sucrose consumption and L-serine production by SYPS-062. In the same time, the metabolic flux distribution was determined in different conditions. The supplementation of folate or VB12 enhanced the cell growth, energy synthesis, and finally increased the flux of pentose phosphate pathway (HMP), whereas the carbon flux to L-serine was decreased. The addition of VB12 not only increased the ratio of L-serine synthesis pathway on G3P joint, but also caused the insufficiency of tricarboxylic acid cycle (TCA) flux, which needed more anaplerotic reaction flux to replenish TCA cycle, that was an important limiting factor for the further increasing of the L-serine productivity.

Phosphoserine aminotransferase deficiency: a novel disorder of the serine biosynthesis pathway.

We present the first two identified cases of phosphoserine aminotransferase deficiency. This disorder of serine biosynthesis has been identified in two siblings who showed low concentrations of serine and glycine in plasma and cerebrospinal fluid. Clinically, the index patient presented with intractable seizures, acquired microcephaly, hypertonia, and psychomotor retardation and died at age 7 mo despite supplementation with serine (500 mg/kg/d) and glycine (200 mg/kg/d) from age 11 wk. The younger sibling received treatment from birth, which led to a normal outcome at age 3 years. Measurement of phosphoserine aminotransferase activity in cultured fibroblasts in the index patient was inconclusive, but mutational analysis revealed compound heterozygosity for two mutations in the PSAT1 gene--one frameshift mutation (c.delG107) and one missense mutation (c.299A-->C [p.Asp100Ala])--in both siblings. Expression studies of the p.Asp100Ala mutant protein revealed a V(max) of only 15% of that of the wild-type protein.

Nac-mediated repression of the serA promoter of Escherichia coli.

Escherichia coli and related bacteria contain two paralogous PII-like proteins involved in nitrogen regulation, the glnB product, PII, and the glnK product, GlnK. Previous studies have shown that cells lacking both PII and GlnK have a severe growth defect on minimal media, resulting from elevated expression of the Ntr regulon. Here, we show that this growth defect is caused by activity of the nac product, Nac, a LysR-type transcription factor that is part of the Ntr regulon. Cells with elevated Ntr expression that also contain a null mutation in nac displayed growth rates on minimal medium similar to the wild type. When expressed from high-copy plasmids, Nac imparts a growth defect to wild-type cells in an expression level-dependent manner. Neither expression of Nac nor lack thereof significantly affected Ntr gene expression, suggesting that the activity of Nac at one or more promoters outside the Ntr regulon was responsible for its effects. The growth defect of cells lacking both PII and GlnK was also eliminated upon supplementation of minimal medium with serine or glycine for solid medium or with serine or glycine and glutamine for liquid medium. These observations suggest that high Nac expression results in a reduction in serine biosynthesis. beta-Galactosidase activity expressed from a Mu d1 insertion in serA was reduced approximately 10-fold in cells with high Nac expression. We hypothesize that one role of Nac is to limit serine biosynthesis as part of a cellular mechanism to reduce metabolism in a co-ordinated manner when cells become starved for nitrogen.

Biosynthesis of sulfur-containing amino acids in Streptomyces venezuelae ISP5230: roles for cystathionine beta-synthase and transsulfuration.

A 0.5 kb fragment of Streptomyces venezuelae ISP5230 genomic DNA was amplified by PCR using primers based on consensus sequences of cysteine synthase isozyme A from bacteria. The deduced amino acid sequence of the PCR product resembled not only cysteine synthase sequences from prokaryotes and eukaryotes but also eukaryotic cystathionine beta-synthase sequences. Probing an Str. venezuelae genomic library with the PCR product located a hybridizing colony from which pJV207 was isolated. Sequencing and analysis of the Str. venezuelae DNA insert in pJV207 detected two ORFs. The deduced amino acid sequence of ORF1 matched both cysteine synthase and cystathionine beta-synthase sequences in GenBank, but its size favoured assignment as a cystathionine beta-synthase. ORF2 in the pJV207 insert was unrelated in function to ORF1; in its sequence the deduced product resembled acetyl-CoA transferases, but disruption of the ORF did not cause a detectable phenotypic change. Disruption of ORF1 failed to elicit cysteine auxotrophy in wild-type Str. venezuelae, but in the cys-28 auxotroph VS263 it prevented restoration of prototrophy with homocysteine or methionine supplements. The change in phenotype implicated loss of the transsulfuration activity that in the wild-type converts these supplements to cysteine. This study concludes that disruption of ORF1 inactivates a cbs gene, the product of which participates in cysteine synthesis by transsulfuration. Enzyme assays of Str. venezuelae mycelial extracts confirmed the formation of cysteine by thiolation of O-acetylserine, providing the first unambiguous detection of this activity in a streptomycete. Enzyme assays also detected cystathionine gamma-synthase, cystathionine beta-lyase and cystathionine gamma-lyase activity in the extracts and showed that the substrate for cystathionine gamma-synthase was O-succinyl-homoserine. Based on assay results, the cys-28 mutation in Str. venezuelae VS263 does not inactivate the cysteine synthase gene but impairs expression in cultures grown in minimal medium.

Role of formaldehyde in direct formation of glycine and serine in bean leaves.

In our early study [2] labelled formaldehyde, glyoxylate, glycine and serine were formed from 14CO2 during 1 min incubation. Continuing the detailed work by unlabelled HCO3- with short (1 min) incubation time in numerous experiments, there were significant changes in the amount of formaldehyde, glyoxylate, glycine and serine. The main product was glycine. In illuminated leaves after HCO3- intake, the formaldehyde content increased. However, in dark in the presence of 5 mM HCO3- the formaldehyde content decreased and the amount of glyoxylate, glycine and serine increased in green leaves. The transamination is improbable as the amount of glutamate and aspartate did not decrease during the glycine and serine formation. The change in the amount of the measured free amino acids and glyoxylate was hindered by (aminooxy)acetic acid (AOA) and amethopterin. AOA, which is an inhibitor of pyridoxal phosphate has no effect on formaldehyde alteration. In the presence of amethopterin which inhibits the activity of tetrahydrofolate the formaldehyde amount increased in dark kept CO2 containing green leaves instead of decreasing. This shows that tetrahydrofolate has a role in glyoxylate formation from formaldehyde and CO2. A direct formation of glycine and serine can be supposed in definite circumstances during photosynthesis, in which pyridoxal phosphate has an important role.

Methionine and serine formation in control and mutant human cultured fibroblasts: evidence for methyl trapping and characterization of remethylation defects.

The conversion of labeled formate to methionine and serine, as a measure of remethylation of homocysteine to methionine and folate coenzyme cycling, has been studied in control and mutant human fibroblasts. Fibroblasts in monolayer culture were incubated with [14C]formate, and labeled methionine sulfone and serine were determined in hydrolysates of oxidized cell proteins. In control cells, methionine and serine were clearly measurable (n = 21, 1.7-5.5 and 2.4-9.7 nmol/mg protein/16 h, respectively). In contrast, methionine formation was reduced in cells from patients with methylenetetrahydrofolate reductase (MR) deficiency (MR mutant, n = 11, 0.05-0.44), combined methylmalonic aciduria/homocystinuria [cobalamin(cbl)C/D mutant, n = 12, 0.014-0.13), and methionine synthase deficiency (MS mutant, n = 3, 0.04-0.23). Furthermore, serine formation was low in cblC/D mutant (0.08-0.98) and MS mutant (0.17-0.94) cells, but normal or high in MR mutant cells (5.2-11.4). Growth of cblC/D mutant cells in medium supplemented with high concentrations of hydroxo-cbl resulted in significant increases of both methionine and serine formation. Taken together these findings provide clear evidence for the existence of the formate to serine pathway described by W. B. Strong and V. Schirch in cultured fibroblasts and indicate that disturbed MS function due to a specific genetic disorder is associated with reduced serine formation in vitro, which reflects availability of reduced folate coenzymes. The correction of this defect by vitamin B12 alone, in cblC/D mutant cell lines, correlates well with the clinical response in the patients and fits in well with the idea that reduced availability of folate coenzymes occurs in functional MS deficiency, in agreement with the methyl trap hypothesis.

Decreased cysteine and proline synthesis in parenterally fed, premature infants.

Little is known about the amino acid (AA) biosynthetic capacity and requirements of premature infants. This study assessed the synthesis of seven biochemically nonessential AA from a universal precursor, glucose, in stable, parenterally fed, premature neonates. Seven infants (six boys, one girl) were studied at a mean age of 6.3 +/- 0.6 (SEM) days; mean gestational age was 29.7 +/- 1.3 (SEM) weeks, and mean birth weight was 1,222.8 +/- 176.5 (SEM) grams. All infants were parenterally fed a mixture of 7.5% to 12.5% dextrose and 2.2% Trophamine, with or without lipid. Mean caloric intake was 93 +/- 8.4 (SEM) kcal/kg/d, and total AA intake was standardized at 2.86 g/kg/d AA, plus supplemental cysteine (30 mg/g AA/d). Each infant received a 4-hour continuous, unprimed intravenous infusion of a stable isotope tracer of D(-)[U13C] glucose (200 mg/kg). Blood samples were obtained before and at the end of the infusion. Conversion of the glucose tracer into seven biochemically nonessential AA (cysteine [Cys], proline [Pro], aspartate [Asp], serine [Ser], glutamate [Glu], alanine [Ala], and glycine [Gly]) was assessed by measuring their isotopic enrichment in plasma, using gas chromatography/mass spectrometry (GC/MS), and expressed as mole percent excess (MPE) (mean +/- SEM). The isotopic enrichment of plasma glucose was also measured using GC/MS. Free plasma AA concentrations (mean +/- SD) were measured using an automated amino acid analyzer. Mean MPE for M + 1, M + 2 and M + 3 Cys, and for M + 1 and M + 3 Pro were not significantly different from 0; M + 2 Pro barely achieved statistical significance (P = .048).(ABSTRACT TRUNCATED AT 250 WORDS)

Consumption and production of amino acids by insulin-responsive cultured fetal rat hepatocytes: the particular case of serine.

The ability for 18-day fetal rat hepatocytes in primary culture to modify extracellular amino acid concentrations was studied between 24 and 48 h of culture. Most of the 19 amino acids tested were found to be taken up by the hepatocytes. However, serine and glutamate appeared in the 24-hour-conditioned medium to be twice as concentrated as in the fresh medium. The profile of net consumption or production of amino acids was unchanged when the medium was supplemented with essential amino acids. The use of [U-14C]glucose revealed that serine released in the medium was mainly formed from glucose. The presence of insulin (10 nM) did neither significantly modify the variations of amino acid concentrations in the medium nor 2-amino[1-14C]isobutyric acid uptake by the cells, while the hormone produced a 2-fold increase in glycogen labeling from [U-14C]glucose. This study revealed that whatever the regulatory culture conditions considered a net serine production out of the cells occurred, which appears to be specific to the fetal stage.

Genetic characterization of a highly efficient alternate pathway of serine biosynthesis in Escherichia coli.

There exists in Escherichia coli a known set of enzymes that were shown to function in an efficient and concerted way to convert threonine to serine. The sequence of reactions catalyzed by these enzymes is designated the Tut cycle (threonine utilization). To demonstrate that the relevant genes and their protein products play essential roles in serine biosynthesis, a number of mutants were analyzed. Strains of E. coli with lesions in serA, serB, serC, or glyA grew readily on minimal medium supplemented with elevated levels of leucine, arginine, lysine, threonine, and methionine. No growth on this medium was observed upon testing double mutants with lesions in one of the known ser genes plus a second lesion in glyA (serine hydroxymethyltransferase), gcv (the glycine cleavage system), or tdh (threonine dehydrogenase). Pseudorevertants of ser mutants capable of growth on either unsupplemented minimal medium or medium supplemented with low levels of leucine, arginine, lysine, threonine, and methionine were isolated. At least two unlinked mutations were associated with such phenotypes.

Effect of adenosine 3',5'-monophosphate on serine metabolism in chick tissues.

The effect of cyclic 3',5'-AMP and supplemental dietary glycine upon de novo synthesis of serine metabolic enzymes in chick livers were examined. Chicks fed crystalline amino acid diets containing 2% glycine had approximately twofold the activity in liver for 3-phosphoglycerate dehydrogenase and phosphoserine phosphatase compared to liver tissue from chicks fed diets lacking in dietary glycine. Chicks subjected to daily intraperitoneal injections of cyclic 3',5'-AMP and fed diets containing no dietary glycine contained biosynthetic enzyme activity similar to glycine-fed chicks suggesting a correlation between glycine and cyclic AMP for serine enzyme induction. The elevated enzyme activity in liver of chicks fed dietary glycine or injected with cyclic AMP was inhibited when chicks were also injected with actinomycin D indicating de novo synthesis of 3-phosphoglycerate dehydrogenase and phosphoserine phosphatase. Dietary glycine or cyclic AMP, however, did not change serine dehydratase and glycerate dehydrogenase activities in chick liver.

Glycine-serine interrelations in the chick.

Glycine and serine are involved in numerous important functions in the body in addition to protein synthesis. Glycine is synthesized by higher animals; however, the rate is not adequate to support maximal growth of the chick. Studies indicate that chicks fed a glycine-serine free crystalline amino acid diet grow at approximately 80% the rate of chicks fed the same diet supplemented with glycine. An equimolar quantity of L-serine has been shown to support equal chick performance as glycine, thus indicating that serine conversion is adequate to meet the dietary need for glycine. The serine-glycine interconversion is catalyzed by the folic acid containing enzyme sering hydroxymethyltransferase, and a deficiency of this vitamin decreases the effectiveness of serine in meeting the chick's dietary need for glycine. Studies with chicks fed normal and high levels of a crystalline amino acid mixture devoid of glycine and serine indicate that the chick has the metabolic potential to synthesize more of these two amino acids than should be required for normal growth. The observed dietary response to glycine or serine or both indicates, however, that this synthetic potential is not being utilized.

Occurrence of phosphorylserine in the spore coat of Bacillus subtilis NRRL B558.

Phosphorylserine was isolated from the spores of Bacillus subtilis NRRL B 558. This phosphorylated amino acid was only found in the spore coat protein and was not detectable in vegetative cells. The compound was characteristically synthesized at a late stage of sporulation and its turnover did not occur until the completion of mature spore formation. The protein which contains phosphorylserine was not solubilized from the spore coat by digestion with pronase or treatment with any detergents.