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1.
Control of meta-cleavage degradation of 4-hydroxyphenylacetate in Pseudomonas putida. 总被引:3,自引:2,他引:1
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Synthesis of enzymes of the 4-hydroxyphenylacetate meta-cleavage pathway was studied in Pseudomonas putida wild-type strain P23X1 (NCIB 9865) and mutant strains which had either structural or regulatory gene mutations. Induction studies with mutant strains each defective in an enzyme of the pathway showed that 4-hydroxyphenylacetate induced the hydroxylase and that 3,4-dihydroxyphenylacetate induced the 2,3-oxygenase, aldehyde dehydrogenase, isomerase, decarboxylase, and hydratase. This showed that the hydroxylase structural gene does not exist in an operon that contains any other structural gene of this meta pathway. Studies of mutant strains that synthesized constitutively the 2,3-oxygenase and subsequent enzymes suggested that the regulation of synthesis of these enzymes was coincident, and, in such strains, the hydroxylase was inducible only. Observations made with a putative polarity mutant that lacked 2,3-oxygenase activity suggested that the structural genes encoding this enzyme and subsequent enzymes of the pathway exist in the same operon. Studies of a regulatory mutant strain that was defective in the induction of the 2,3-oxygenase and subsequent enzymes suggest that the 2,3-oxygenase operon is under positive control. 相似文献
2.
Immunochemical characterization of glutamine synthetase from Neurospora crassa glutamine auxotrophs.
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Glutamine synthetase derived from two Neurospora crassa glutamine auxotrophs was characterized. Previous genetic studies indicated that the mutations responsible for the glutamine auxotrophy are allelic and map in chromosome V. When measured in crude extracts, both mutant strains had lower glutamine synthetase specific activity than that found in the wild-type strain. The enzyme from both auxotrophs and the wild-type strain was partially purified from cultures grown on glutamine as the sole nitrogen source, and immunochemical studies were performed in crude extracts and purified fractions. Quantitative rocket immunoelectrophoresis indicated that the activity per enzyme molecule is lower in the mutants than in the wild-type strain; immunoelectrophoresis and immunochemical titration of enzyme activity demonstrated structural differences between the enzymes from both auxotrophs. On the other hand, the monomer of glutamine synthetase of both mutants was found to be of a molecular weight similar to that of the wild-type strain. These data indicate that the mutations are located in the structural gene of N. crassa glutamine synthetase. 相似文献
3.
Purification and in vitro complementation of mutant histidinol dehydrogenases. 总被引:3,自引:0,他引:3
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The biochemistry of interallelic complementation within the Salmonella typhimurium hisD gene was investigated by in vitro protein complementation of mutant histidinol dehydrogenases (EC 1.1.1.23). Double-mutant strains were constructed containing the hisO1242 (constitutive overproducer) attenuator mutation and selected hisDa or hisDb mutations. Extracts from such hisDa986 and hisDb1799 mutant cells failed to show histidinol dehydrogenase activity but complemented to produce active enzyme. Inactive mutant histidinol dehydrogenases were purified from each of the two mutants by ion-exchange chromatography. Complementation by the purified mutant proteins required the presence of 2-mercaptoethanol and MnCl2, and protein-protein titrations indicated that heterodimers were strongly preferred in mixtures of the complementary mutant enzymes. Neither mutant protein showed negative complementation with wild-type enzyme. The Vmax for hybrid histidinol dehydrogenase was 11% of that for native enzyme, with only minor changes in Km values for substrate or coenzyme. Both purified mutant proteins failed to catalyze NAD-NADH exchange reactions reflective of the first catalytic step of the two-step reaction. The inactive enzymes bound 54Mn2+ weakly or not at all in the presence of 2-mercaptoethanol, in contrast to wild-type enzyme which bound 54Mn2+ to 0.6 sites per monomer under the same conditions. The mutant proteins, like wild-type histidinol dehydrogenase, behaved as dimers on analytical gel filtration chromatography, but dissociated to form monomers in the presence of 2-mercaptoethanol. This effect of 2-mercaptoethanol was prevented by low levels of MnCl2. It thus appears that mutant histidinol dehydrogenase molecules bind metal ion poorly. The complementation procedure may allow for formation of a functional Mn2+-binding site, perhaps at the subunit interface. 相似文献
4.
Effects of Mutations and Growth Conditions on Lipid Synthesis in Neurospora crassa 总被引:3,自引:1,他引:2
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A morphological mutant (col-2) of Neurospora, which is partially deficient in glucose-6-phosphate dehydrogenase (G-6-PD) activity and has lower levels of reduced nicotinamide adenine dinucleotide phosphate (NADPH), accumulated three-fold more triglycerides during log-phase growth than the wild-type strain. Increased lipid deposition was not found in other strains that included slow-growing morphological mutants, NADPH-deficient strains, G-6-PD-deficient mutants, wild-type revertants from col-2, and a cel, col-2 double mutant. The cel, col-2 strain was supplemented with an exogenous source of fatty acids because it cannot synthesize these lipid moieties. The observed normal lipid content of this strain suggests that the lipid deposition in col-2 on glucose is due to an overstimulation of fatty acid synthesis and not a deficiency in fatty acid breakdown. The neutral lipid levels in both wild type and col-2 were decreased to identical levels when grown on glutamate as a carbon source. This effect was not due to changes in glutamic dehydrogenase levels. The omission of citrate from the glutamate medium reduced wild-type neutral lipid levels even further, but had no effect on col-2. The variations with time in the neutral lipid levels of col-2 upon changes in these carbon sources are presented, as well as a discussion of the possible types of regulatory effects unique to the col-2 mutation which might affect fatty acid synthesis. 相似文献
5.
Glucose-6-phosphate dehydrogenase has been purified to near homogeneity from wild-type Escherichia coli K-12 and from a mutant previously found to contain substantially more of the enzyme. The two enzymes are the same in all characteristics studied thus far: specific activity, kinetics, specificity, and subunit size. 相似文献
6.
Isolation of a gene for a regulatory 15-kDa subunit of mitochondrial F1F0-ATPase and construction of mutant yeast lacking the protein 总被引:1,自引:0,他引:1
Y Yoshida T Sato T Hashimoto N Ichikawa S Nakai H Yoshikawa F Imamoto K Tagawa 《European journal of biochemistry》1990,192(1):49-53
A gene coding for yeast 15-kDa protein, a regulatory factor of mitochondrial F1F0-ATPase, was isolated. The cloned gene was disrupted in vitro and mutant strains that did not contain the 15-kDa protein were constructed by transformation of yeast cells with the disrupted gene. The ATP-synthesizing activity of the mutant mitochondria was the same as that of wild-type cells, suggesting that the 15-kDa protein is not required for mitochondrial oxidative phosphorylation. Collapse of the membrane potential induced ATP-hydrolyzing activity of F1F0-ATPase of the mutant mitochondria but not of normal mitochondria. Activation of the enzyme was also observed during incubation of submitochondrial particles from mutant cells, but not of those from wild-type cells. Thus, it is inferred that the 15-kDa protein supports the action of an intrinsic ATPase inhibitor of the ATP-hydrolyzing activity of the enzyme upon de-energization of mitochondrial membranes. 相似文献
7.
tRNA nucleotidyltransferase is not essential for Escherichia coli viability. 总被引:9,自引:1,他引:8
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The role of tRNA nucleotidyltransferase in Escherichia coli has been uncertain because all tRNA genes studied in this organism already encode the -C-C-A sequence. Examination of a cca mutant, originally thought to contain 1-2% enzyme activity, indicated that it actually produces an inactive fragment of 40 kd compared to 47 kd for the wild-type enzyme due to a nonsense mutation in its cca gene. To confirm that the residual activity in extracts of this strain is due to another enzyme, and that tRNA nucleotidyltransferase is non-essential, we have interrupted the cca gene in vitro, and transferred this mutant gene to a variety of strains. In all cases mutant strains are viable, although as much as 15% of the tRNA population contains defective 3' termini, and no tRNA nucleotidyltransferase is detectable. Mutant strains grow slowly, but can be restored to more normal growth by a relA mutation or by a decrease in RNase T activity. In the latter case the amount of defective tRNA decreases dramatically. These findings indicate that tRNA nucleotidyltransferase is not essential for E. coli viability, and therefore, that all essential tRNA genes in this organism encode the -C-C-A sequence. 相似文献
8.
Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution.
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S. Akanuma A. Yamagishi N. Tanaka T. Oshima 《Protein science : a publication of the Protein Society》1998,7(3):698-705
We improved the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by an in vivo evolutionary technique using an extreme thermophile, Thermus thermophilus, as a host cell. The leuB gene encoding B. subtilis 3-isopropylmalate dehydrogenase was integrated into the chromosome of a leuB-deficient strain of T. thermophilus. The resulting transformant showed a leucine-autotrophy at 56 degrees C but not at 61 degrees C and above. Phenotypically thermostabilized strains that can grow at 61 degrees C without leucine were isolated from spontaneous mutants. Screening temperature was stepwise increased from 61 to 66 and then to 70 degrees C and mutants that showed a leucine-autotrophic growth at 70 degrees C were obtained. DNA sequence analyses of the leuB genes from the mutant strains revealed three stepwise amino acid replacements, threonine-308 to isoleucine, isoleucine-95 to leucine, and methionine-292 to isoleucine. The mutant enzymes with these amino acid replacements were more stable against heat treatment than the wild-type enzyme. Furthermore, the triple-mutant enzyme showed significantly higher specific activity than that of the wild-type enzyme. 相似文献
9.
Effect of mutations in the qa gene cluster of Neurospora crassa on the enzyme catabolic dehydroquinase. 总被引:1,自引:1,他引:0
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Catabolic dehydroquinase, which functions in the inducible quinic acid catabolic pathway of Neurospora crassa, has been purified from wild type (74-A) and three mutants in the qa gene cluster. The mutant strains were: 105c, a temperature-sensitive constitutive mutant in the qa-1 regulatory locus; M-16, a qa-3 mutant deficient in quinate dehydrogenase activity; and 237, a leaky qa-2 mutant which possess very low levels of catabolic dehydroquinase activity. The enzymes purified from strains 74-A, 105c, and M-16 are identical with respect to behavior during purification, specific activity, electrophoretic behavior, stability, molecular weight, subunit structure, immunological cross-reactivity, and amino acid content. The mutant enzyme from strain 237 is 1,500-fold less active and appears to have a slightly different amino acid content. It is identical by a number of the other criteria listed above and is presumed to be a mutant at or near the enzyme active site. These data demonstrate that the qa-1 gene product is not involved in the posttranslational expression of enzyme activity. The biochemical identity of catabolic dehydroquinase isolated from strains 105c and M-16 with that from wild type also demonstrates that neither the inducer, quinic acid, nor other enzymes encoded in the qa gene cluster are necessary for the expression of activity. Therefore the combined genetic and biochemical data on the qa system continue to support the hypothesis that the qa-1 regulatory protein acts as a positive initiator of qa enzyme synthesis. 相似文献
10.
Biochemical identification and phylogenetic relationships in free-living amoebas of the genus Naegleria 总被引:1,自引:0,他引:1
Using isoelectric focusing, the zymograms of 23 pathogenic and nonpathogenic Naegleria strains were studied for the activity of 16 enzymes. Certain enzymes (lactate dehydrogenase, L-threonine dehydrogenase, superoxide dismutase, acid phosphatase, malic enzyme, and leucine aminopeptidase) proved particularly useful from a practical point of view as they allow easy and reliable identification of pathogenic N. fowleri and N. australiensis as well as nonpathogenic N. lovaniensis strains. Genetic interpretation of these zymograms gave estimates of genetic distances that largely confirmed the taxonomic position of the Naegleria species. In addition, the genetic data suggest that there are two main phylogenetic groups in the genus Naegleria. 相似文献
11.
31株苏云金芽孢杆菌杀虫晶体蛋白基因型鉴定及表达产物研究 总被引:9,自引:1,他引:8
利用已建立的苏云金芽孢杆菌cry基因的PCRRFLP鉴定体系,鉴定了31株Bt菌株的cry基因类型,并进行了SDSPAGE分析和杀虫生物活性测定。研究表明:25株含cry1基因,表达蛋白130~150kD;其中16株含有对鞘翅目和鳞翅目害虫皆有活性的cry1I基因,其表达蛋白为81kD;15株同时含有cry1和cry2基因(13株表达蛋白约为60kD);10株含有未知待定基因;6株不含所鉴定的cry基因(其中2株有表达产物)。室内生物测定表明:cry1、cry2基因表达的菌株对鳞翅目害虫具有高杀虫活性,7株对舞毒蛾和膜翅目——杨叶蜂幼虫具有较高杀虫活性;含有cry1Aa\,cry1Ac\,cry2或cry1Ab\,cry1Ac\,cry2基因组合的菌株对棉铃虫幼虫均显示杀虫活性,其中6、12、30号菌株毒力最强。不含上述cry基因的菌株均无杀虫活性。以上结果证明,通过cry基因类型鉴定和表达产物的SDSPAGE分析可以预测菌株的杀虫活性。 相似文献
12.
Alcohol dehydrogenase (EC 1.1.1.1) activity was measured in liver extracts from one outbred and three inbred strains of rats. Strain-specific differences in enzyme activity were observed in the adult male rats. The differences appeared as the animals reached puberty. Studies on the enzyme purified from Sprague-Dawley and ACI rats indicate that the enzymes in these strains are identical and that the difference in activity found in liver extracts is due to differences in the amount of enzyme present. Genetic crosses between Sprague-Dawley and ACI rats suggest that the liver content of alcohol dehydrogenase is controlled by an autosomal regulatory locus with the characteristics of a temporal gene. 相似文献
13.
Plasmid-determined alcohol dehydrogenase activity in alkane-utilizing strains of Pseudomonas putida. 总被引:10,自引:9,他引:1
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We have identified an alcohol dehydrogenase activity in Pseudomonas putida strains carrying the CAM-OCT degradative plasmid that were grown on octane. The activity is nicotinamide adenine dinucleotide independent, sediments at 48,000 x g, and shows 20-fold greater activity with octanol rather than butanol as substrate. The enzyme is inducible by unoxidized alkane and is present only in strains that have the OCT plasmid genes for alkane degradation with a wild-type alcO locus. No analogous chromosomal dehydrogenase could be detected. Wild-type and actanol-negative mutants (alcA-) without plasmids both contain a constitutive nicotinamide adenine dinucleotide-linked soluble alcohol dehydrogenase activity. This means that alcA- mutants are cryptic for octanol oxidation and suggests that the particulate plasmid-coded alcohol dehydrogenase activity is active on surface- or membrane-bound substrate. 相似文献
14.
Mutations affecting glutamine synthetase activity in Salmonella typhimurium. 总被引:18,自引:12,他引:6
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A positive selection procedure has been devised for isolating mutant strains of Salmonella typhimurium with altered glutamine synthetase activity. Mutants are derived from a histidine auxotroph by selecting for ability to grow on D-histidine as the sole histidine source. We hypothesize that the phenotype may be based on a regulatory increase in the activities of the D-histidine racemizing enzymes, but this has not been established. Spontaneous glutamine-requiring mutants isolated by the above selection procedure have two types of alterations in glutamine synthetase activity. Some have less than 10% of parent activity. Others have significant glutamine synthetase activity, but the enzyme have an altered response to divalent cations. Activity in mutants of the second type mimics that of highly adenylylated wild-type enzyme, which is believed to be in-active in vivo. Glutamine synthetase from one such mutant is more heat labile than wild-type enzyme, indicating that it is structurally altered. Mutations in all strains are probably in the glutamine synthetase structural gene (glnA). They are closely linked on the Salmonella chromosome and lie at about min 125. The mutants have normal glutamate dehydrogenase activity. 相似文献
15.
Characterization of the carbohydrate component of fraction I in the Neurospora crassa cell wall.
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The carbohydrate portion of fraction I of the Neurospora crassa cell wall has been analyzed for sugar composition by gas-liquid chromatography and colorimetric methods. The analysis was performed comparatively in a wild-type strain (RL 3-8A) and three morphological mutants: scumbo (FGSC 49), peak-2a (a mutant known to be allelic to biscuit), and ragged (FGSC 296). Fraction I of all strains studied contains glucose, mannose, and galactose as the main sugars. Uronic acids and amino sugars are also present in small amounts. The glycosidic linkages binding the neutral sugars were analyzed by Lindberg's combined gas chromatography-mass spectrometry techniques for identification of the partially methylated alditol acitate sugar derivatives. The main polymeric portion of fraction I seems to be a linear glucan with the glucose residues linked by 1 leads to 3 and 1 leads to 4 bonds. A mannan portion with a branched configuration is also present, with galactose as the sugar residue which serves as branches in the molecule(s). The branched mannan portion appears to increase in amount in correlation with more drastic morphological changes of the mycelia. In this respect, the mutant ragged has the lowest mycelial growth rate and the largest amount of mannan. The importance of the polysaccharide structure of fraction I on the colonial morphology of the mycelia is discussed. 相似文献
16.
One-carbon metabolism in Neurospora crassa wild-type and in mutants partially deficient in serine hydroxymethyltransferase.
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1. The concentrations of folate-dependent enzymes in Neurospora crassa Lindegren A wild type (FGSC no. 853), Ser-l mutant, strain H605a (FGSC no. 118), and for mutant, strain C-24 (FGSC no. 9), were compared during exponential growth on defined minimal media. Both mutants were partially lacking in serine hydroxymethyltransferase, but contained higher concentrations of 10-formyltetrahydrofolate synthetase than did the wild type. Mycelia of the mutants contained higher concentrations of these enzymes when growth media were supplemented with 1mM-glycine. In the wild-type, this glycine supplement also increased the specific activities of 5,10-methylenetetrahydrofolate dehydrogenase and 5,10-methylenetetrahydrofolate reductase. 5. During growth, total folate and polyglutamyl folate concentrations were greatest in the wild-type. Methylfolates were not detected in mutant Ser-l, and were only present in the for mutant after growth in glycine-supplemented media. Exogenous glycine increased folate concentration threefold in the wild type, mainly owing to increases in unsubstituted polyglutamyl derivatives. 3. Feeding experiments using 14C-labelled substrates showed that C1 units were generated from formate, glycine and serine in the wild type. Greater incorporation of 14C occurred when mycelia were cultured in glycine-supplemented media. Formate and serine were precursors of C1 units in the mutants, but the ability to cleave glycine was slight or lacking. 相似文献
17.
Ching T. Hou Ramesh N. Patel Allen I. Laskin Nancy Barnabe 《Archives of biochemistry and biophysics》1982,216(1):296-305
An NAD-linked formate dehydrogenase (EC 1.2.1.2.) from methanol-grown Pichia pastoris NRRL Y-7556 has been purified. The purification procedure involved ammonium sulfate fractionation, hollow-fiber H1P10 filtration, ion-exchange chromatography, and gel filtration. Both dithiothreitol (10 mm) and glycerol (10%) were required for stability of the enzyme during purification. The final enzyme preparation was homogeneous as judged by polyacrylamide gel electrophoresis and by sedimentation pattern in an ultracentrifuge. The enzyme has a molecular weight of 94,000 and consists of two subunits of identical molecular weight. Formate dehydrogenase catalyzes specifically the oxidation of formate. No other compounds tested can replace NAD as the electron acceptor. The Michaelis constants were 0.14 mm for NAD and 16 mm for formate (pH 7.0, 25 °C). Optimum pH and temperature for formate dehydrogenase activity were around 6.5–7.5 and 20–25 °C, respectively. Amino acid composition of the enzyme was also studied. Antisera prepared against the purified enzyme from P. pastoris NRRL Y-7556 form precipitin bands with isofunctional enzymes from different strains of methanol-grown yeasts, but not bacteria, on immunodiffusion plates. Immunoglobulin fraction prepared against the enzyme from yeast strain Y-7556 inhibits the catalytic activity of the isofunctional enzymes from different strains of methanol-grown yeasts. 相似文献
18.
《Bioscience, biotechnology, and biochemistry》2013,77(8):1524-1532
We report the molecular characterization and physiological function of a novel L-aspartate dehydrogenase (AspDH). The purified enzyme was a 28-kDa dimeric protein, exhibiting high catalytic activity for L-aspartate (L-Asp) oxidation using NAD and/or NADP as cofactors. Quantitative real-time PCR analysis indicated that the genes involved in the AspDH gene cluster, poly-3-hydroxyalkanoate (PHA) biosynthesis, and the TCA cycle were substantially induced by L-Asp in wild-type cells. In contrast, expression of the aspartase and aspartate aminotransferase genes was substantially induced in the AspDH gene knockout mutant (ΔB3576) but not in the wild type. GC-MS analyses revealed that the wild-type strain synthesized poly-3-hydroxybutyrate from fructose or L-Asp, whereas the ΔB3576 mutant did not synthesize PHA from L-Asp. AspDH gene cluster products might be involved in the biosynthesis of the PHA precursor, revealing that AspDH was a non-NadB type enzyme, and thus entirely different from the previously reported NadB type enzymes working in NAD biosynthesis. 相似文献
19.
Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli 总被引:1,自引:0,他引:1
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Jeffrey D. Hillman 《The Biochemical journal》1979,179(1):99-107
NAD+-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD+ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD+ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme. 相似文献
20.
Serine hydroxymethyltransferase (SHMT), commonly implicatedin the glycine synthesis of eucaryotes, was examined in Neurosporacrassa, wild type (FGSC 853) and a formate-requiring mutant(FGSC 9). The mutant was SHMT-deficient, containing only 15%of the total activity found in the wild type. Differential anddensity gradient centrifugations showed the mutant to be deficientin soluble SHMT activity. Both strains contained particulateSHMT which sedimented with mitochondrial marker enzymes. The origins of glycine were examined by a combination of enzyme,growth and 14C feeding experiments. Growth of the mutant wasstrongly inhibited by the isocitrate lyase-directed inhibitoritaconate. This inhibition was reduced when exogenous glycinewas supplied. Itaconate (up to 30 mM) did not inhibit growthof the wild type but in both strains isocitrate lyase activitieswere reduced. The mutant contained more lyase and glyoxylateaminotransferase than the wild type. In feeding experiments,[2-14C]acetate and [l-14C]glyoxylate were more readily incorporatedinto glycine in the mutant than the wild type. Itaconate (30mM) reduced the flow of acetate carbon into glycine by up to70% in the mutant. It is concluded that deficiency in solubleSHMT necessitates glycine synthesis via an isocitrateglyoxylateglycinesequence. (Received December 24, 1979; ) 相似文献