delta-Aminolevulinic acid dehydratase activity in erythrocytes of diabetic patients

Erythrocytes delta-aminolevulinate dehydratase activity was assayed in 41 diabetic patients and 33 normal controls. It was found that in diabetic patients the erythrocyte ALA-D activity was lower than in controls, and the difference of the mean values was statistically highly significant (P less than 0.001). We found a significant negative correlation (r = - 0.846, P less than 0.001) between ALA-D activity and blood glucose levels. For this reason, using normal adult human whole blood haemolysates, it was investigated the effects in vitro of glucose and insulin on normal erythrocytic ALA-D. No significant difference in ALA-D activity was found in the presence of insulin. On the other hand, there was considerable decrease in the enzyme activity in the blood samples after glucose addition.     

Message amplification phenotyping of an inherited delta-aminolevulinate dehydratase deficiency in a family with acute hepatic porphyria

The molecular basis of the enzymatic defect responsible for acute hepatic porphyria due to delta-aminolevulinate dehydratase (ALAD) deficiency was investigated in a family including a proband with the acute disease. In order to delineate the mutation in the proband, cDNA for deficient ALAD was synthesized from the proband's cells. The ALAD phenotype was studied by message amplification phenotyping with total RNA extracted from lymphoblastoid cells of the proband and his family members. Two independent mutant alleles of ALAD were identified in the proband's cells. One mutant allele was shown to result in an amino acid substitution at residue 274 (Ala274----Thr). Message amplification phenotyping studies have also permitted us to define the ALAD phenotype of each subject in the family. This is the first mutation to be recognized in the human ALAD gene.     

Identification of the enzymatic basis for delta-aminolevulinic acid auxotrophy in a hemA mutant of Escherichia coli.

The hemA mutation of Escherichia coli K-12 confers a requirement for delta-aminolevulinic acid (ALA). Cell extract prepared from the hemA strain SASX41B was incapable of producing ALA from either glutamate or glutamyl-tRNA, whereas extract of the hem+ strain HB101 formed colorimetrically detectable amounts of ALA and transferred label from 1-[14C]glutamate and 3,4-[3H]glutamyl-tRNA to ALA. Extracts of both strains converted glutamate-1-semialdehyde to ALA and were capable of aminoacylating tRNAGlu. Glutamyl-tRNA formed by extracts of both strains could be converted to ALA by the extract of hem+ cells. The extract of hemA cells did not convert glutamyl-tRNA formed by either strain to ALA. However, the hemA cell extract, when supplemented in vitro with glutamyl-tRNA dehydrogenase isolated from Chlorella vulgaris cells, formed about as much ALA as did the unsupplemented hem+ cell extract. We conclude from these observations that the enzyme activity that is lacking in the ALA auxotrophic strain carrying the hemA mutation is that of glutamyl-tRNA dehydrogenase.     

Crystal structure of D-serine dehydratase from Escherichia coli

D-Serine dehydratase from Escherichia coli is a member of the β-family (fold-type II) of the pyridoxal 5'-phosphate-dependent enzymes, catalyzing the conversion of D-serine to pyruvate and ammonia. The crystal structure of monomeric D-serine dehydratase has been solved to 1.97?-resolution for an orthorhombic data set by molecular replacement. In addition, the structure was refined in a monoclinic data set to 1.55? resolution. The structure of DSD reveals a larger pyridoxal 5'-phosphate-binding domain and a smaller domain. The active site of DSD is very similar to those of the other members of the β-family. Lys118 forms the Schiff base to PLP, the cofactor phosphate group is liganded to a tetraglycine cluster Gly279-Gly283, and the 3-hydroxyl group of PLP is liganded to Asn170 and N1 to Thr424, respectively. In the closed conformation the movement of the small domain blocks the entrance to active site of DSD. The domain movement plays an important role in the formation of the substrate recognition site and the catalysis of the enzyme. Modeling of D-serine into the active site of DSD suggests that the hydroxyl group of D-serine is coordinated to the carboxyl group of Asp238. The carboxyl oxygen of D-serine is coordinated to the hydroxyl group of Ser167 and the amide group of Leu171 (O1), whereas the O2 of the carboxyl group of D-serine is hydrogen-bonded to the hydroxyl group of Ser167 and the amide group of Thr168. A catalytic mechanism very similar to that proposed for L-serine dehydratase is discussed.     

Inherited deficiency of delta-aminolevulinic acid dehydratase.

Delta-aminolevulinic acid dehydratase (ALA-D) is the second enzyme in the porphyrin-heme pathway and converts delta-aminolevulinc acid (ALA) to porphobilinogen (PBG). A family is reported with an inherited deficiency of red cell ALA-D activity occurring over three generations in an autosomal dominant pattern. Intial experiments support the hypothesis that the mutation in this family may affect a regulatory gene, but enzyme purification and further study are required. Although no clinical manifestations of deficient ALA-D activity have been found in affected persons, families such as this may be at increased risk for the serious consequences of lead poisoning, which produces marked inhibition of ALA-D activity.     

A selective inhibitor of Escherichia coli prephenate dehydratase.

To identify selective prephenate dehydratase (PDT) inhibitors, a series of substituted biphenic acid derivatives was synthesized using the Ullmann reaction. Screening experiments identified 18 as a promising new PDT inhibitor.     

Ubiquinone deficiency in an auxotroph of Escherichia coli requiring 4-hydroxybenzoic acid

1. Escherichia coli 156:53D2 synthesized ubiquinone only when the growth medium was supplemented with 4-hydroxybenzoate acid. 2. Little or no vitamin K(2) was formed by the mutant under the growth conditions employed, in contrast with wild-type strains. 3. In the mutant ubiquinone deficiency was correlated with low respiration and with low particulate NADH-oxidase and NADH-cytochrome b(1)-reductase activity. 4. Preincubation of ubiquinone-deficient particles with ubiquinone-30 largely restored the NADH-oxidase and NADH-cytochrome b(1)-reductase activities. 5. Various NADH-dye-linked reductases which may be associated with NADH dehydrogenase were not affected by the absence of ubiquinone. 6. The succinate-oxidase complex was less affected than the particulate NADH oxidase by ubiquinone deficiency. 7. A pathway for electrons in the NADH-oxidase complex of the auxotroph of E. coli is proposed and its relationship to the pathway in the wild-type strain is discussed.     

Altered expression of biodegradative threonine dehydratase in Escherichia coli mutants.

A number of strains of Escherichia coli K-12 failed to synthesize significant amounts of biodegradative threonine dehydratase (EC 4.2.1.16) when grown anaerobically in tryptone-yeast extract medium, a condition which is optimal for the induction of this enzyme. However, the addition of 10 mM potassium nitrate to the culture medium enabled a few of these strains, notably MB201, to induce the enzyme. An examination of the kinetic parameters, modifier sensitivity, and immunological cross-reactivity revealed that the enzyme produced by MB201 in nitrate-supplemented medium appeared indistinguishable from the dehydratase of a wild-type strain. The reduced expression of threonine dehydratase in MB201 appeared highly specific; the synthesis of two other inducible enzymes, D-serine deaminase and tryptophanase, and two "anaerobic" proteins, namely, fumarate reductase and cytochrome c551, remained unaffected. The mutation (tdcI) responsible for the altered expression of the dehydratase in MB201 was located at min 91 on the E. coli chromosome and appeared to tightly linked to if not identical with pgi, the gene encoding phosphoglucose isomerase, as judged by growth experiments on glucose and fructose, direct assay of phosphoglucose isomerase activity, spontaneous and simultaneous reversion of MB201 (tdcI) to TdcI+ and Pgi+ phenotype, and cosegregation of the two loci during transduction with P1 phage. Because not all strains lacking the dehydratase showed nitrate-dependent enzyme synthesis or had lesions at the pgi locus, it appears that mutations at multiple loci on the E. coli chromosome may influence the expression of the enzyme in vivo.     

Amino acid sequence of the regulatory-site glyoxylate peptide of biodegradative threonine dehydratase of Escherichia coli.

Incubation of purified Escherichia coli biodegradative threonine dehydratase with glyoxylate resulted in covalent binding of 1 mol of glyoxylate per mol of protein with concomitant loss of enzyme activity. The glyoxylate-binding site was identified as a heptapeptide representing amino acid residues Ser-33-Asn-Tyr-Phe-Ser-Glu-Arg-39 in the protein primary structure. Addition of glyoxylate to a culture of E. coli cells led to time-dependent enzyme inactivation. Immunoprecipitation with anti-dehydratase antibody of extract from [14C]glyoxylate-treated cells revealed labeled dehydratase polypeptide. These results are interpreted to mean that enzyme inactivation by glyoxylate in E. coli cells is associated with covalent protein modification.     

Superoxide sensitivity of the Escherichia coli 6-phosphogluconate dehydratase.

The activity of 6-phosphogluconate dehydratase was significantly lower in extracts of aerobically grown Escherichia coli deficient in superoxide dismutase (sodAsodB) and in mutants lacking the inducible manganese-containing superoxide dismutase (sodA), exposed to the redox-cycling agent paraquat, than in the parental strain. Growth of these strains on a gluconate minimal medium was also impaired under these conditions. The enzyme was most susceptible to dioxygen in superoxide dismutase (SOD)-free extracts, and exogenous SOD afforded a concentration-dependent protection against inactivation. The amount of SOD necessary for full protection was comparable to the amount normally present in extracts of aerobic E. coli (7-36 units/mg protein), and the rate of reaction of O2- with the dehydratase was estimated to be approximately 2.0 x 10(8) M-1 s-1. The dehydratase was much less sensitive to O2 or H2O2 than to O2-. The virtual substrate, alpha-glycerophosphate, provided partial protection. Iron chelators, thiol-reactive reagents, and oxidants, including nitrite and diamide, inactivated the enzyme. Fluoride ions stabilized the dehydratase and blocked the effect of oxidants. The O2(-)-sensitive target site is proposed to be an iron-sulfur cluster which is readily destroyed by oxidation.     

Catabolite inactivation of biodegradative threonine dehydratase of Escherichia coli.

Incubation of Escherichia coli cells with glucose, pyruvate, and certain other metabolites led to rapid inactivation of inducible biodegradative threonine dehydratase. Analysis with several mutant strains showed that pyruvate, and not a metabolite derived from pyruvate, was capable of inactivating enzyme, and that glucose acted indirectly after being converted to pyruvate. Some other alpha-keto acids such as oxaloacetate and alpha-ketobutyrate (but not alpha-ketoglutarate) were also effective. Inactivation of threonine dehydratase by pyruvate was also observed with purified enzyme preparations. The rates of enzyme inactivation increased with increased concentrations of pyruvate and decreased with increased levels of AMP. Increasing protein concentrations lowered the rates of enzyme inactivation. Dithiothreitol had a large effect on the maximum extent of inactivation of the enzyme by pyruvate; high concentrations of AMP and DTT almost completely counteracted the effect of pyruvate. Gel filtration data showed that pyruvate influenced the oligomeric state of the enzyme by altering the association-dissociation equilibrium in favor of dissociation; the Stokes' radius of the pyruvate-inactivated enzyme was 32 A as compared to 42 A for the untreated enzyme. Reassociation of the dissociated form of the enzyme was achieved by removal of excess free pyruvate by dialysis against buffer supplemented with AMP and DTT. Incubation of threonine dehydratase with [14-C]pyruvate revealed apparent covalent attachment of pyruvate to the enzyme. Strong protein denaturants such as guanidine, urea, and sodium dodecyl sulfate failed to release bound radioactive pyruvate; the molar ratio of firmly bound pyruvate was approximately 1 mol/150,000 g of protein. Pretreatment of the enzyme with p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) did not reduce the binding of [14-C]pyruvate suggesting no active site SH was involved in the pyruvate-enzyme linkage. Titration of active and pyruvate-inactivated enzyme with Nbs2 indicated that the loss in enzyme activity was not due to oxidation of essential sulfhydryl groups on the enzyme. Based on these data we propose that the mechanism of enzyme inactivation by pyruvate involves covalent attachment of pyruvate to the active oligomeric form of the enzyme followed by dissociation of the oligomer to yield inactive enzyme.     

Decrease of hepatic delta-aminolevulinate dehydratase activity in an animal model of fatigue

Fatigue can be defined physiologically as inability to maintain the expected power output. At present, no standard of fatigue are yet available. In order to find biomarkers of fatigue, we investigated the level of delta-aminolevulinic acid (ALA), the first intermediate metabolite in the heme biosynthetic pathway, in the plasma and urine of an animal model of fatigue. To prepare fatigued animals, we kept rats for 5 days in a cage filled with water to a height of 1.5 cm. As a result, the plasma and urinary ALA levels were increased in the fatigued animals as compared with those in the control animals. One day after the rats had been returned to their normal cages, these increased levels were restored to the control ones. We also examined the activity of the enzyme ALA dehydratase (ALAD), which is the second enzyme in the heme biosynthetic pathway, and ALAD gene expression during the fatigue and its recovery sessions. The ALAD activity, as well as its gene expression, in the liver of the fatigued animals was decreased as compared with those of the control animals. Both activity and gene expression of ALAD were recovered to their respective control levels after the rats had been allowed to rest in their normal cages for 1 day. Furthermore, the activity of ALA synthase (ALAS), the rate-limiting enzyme in the heme biosynthesis, in the liver was increased after the fatigue session for 5 days. Although this level of increase in the plasma concentration of ALA may not induce fatigue, increase in plasma and urinary ALA levels can be biomarkers of fatigue.     

Requirements for induction of the biodegradative threonine dehydratase in Escherichia coli.

Synthesis of the biodegradative L-threonine dehydratase in Escherichia coli, Crookes strain, was prevented by dissolved oxygen concentrations of 6 micrometer or greater. This effect was shown to be exerted solely on synthesis, rather than being the result of enzyme inactivation in vivo. In addition to an anaerobic environment, maximum enzyme synthesis was dependent upon the presence of a complete complement of amino acids, with omission of L-threonine, L-valine, or L-leucine producing the largest decreases in enzyme formation. L-Threonine, the most essential of the amino acid requirements, could be partially replaced by DL-allothreonine or alpha-ketobutyrate. Half-maximal stimulation of enzyme synthesis occurred with 0.4 mM threonine in the medium. The roles of anaerobiosis and amino acids are interpreted as being in accord with the concept that threonine dehydratase functions in anaerobic energy production under conditions of amino acid sufficiency.     

Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli.

Escherichia coli K-12 has two genes, asnA+ and asnB+, either one of which is able to satisfy the need of cells for asparagine. In order for a strain to have an auxotrophic requirement for asparagine, both genes must be mutationally inactivated. We obtained mutants with Tn5 inserted in asnB. asnB was mapped by conjugation and by three-factor P1 transductions at 15 min on the E coli K-12 linkage map, between ubiF and nagB. Specialized transducing phage lamba 781 supE was shown to carry asnB, as well as supE, ubiF, nagA, and nagB. asnA is the previously mapped ilv-linked asn locus, whiich is between uncB and rbs. E. coli C also has two asn genes, corresponding to asnA and asnB.     

Experimental hypothyroidism inhibits delta-aminolevulinate dehydratase activity in neonatal rat blood and liver

The aim of this study was to investigate the potential relationship between hypothyroidism and delta-aminolevulinate dehydratase (delta-ALA-D) activity in rat blood and liver. Experimental hypothyroidism was induced in weanling rats by exposing their mothers to propylthiouracil (PTU) diluted in tap water (0.05% w/ v), ad libitum, during the lactational period (PTU group). Control (euthyroid) group included weanling rats whose mothers received just tap water, ad libitum, during the lactational period. Reverted-hypothyroid group (PTU + 3,3',5-triiodo-L-thyronine [T(3)]) included weanling rats whose mothers were exposed to PTU similarly to those in the hypothyroid group, but pups received daily subcutaneous injections of T(3) (20 microg/kg, from Postnatal Days 2-20). After the treatment, serum T(3) levels were drastically decreased (around 70%) in the PTU group, and this phenomenon was almost reverted by exogenous T(3). PTU decreased blood delta-ALA-D activity by 75%, and T(3) treatment prevented such phenomena. Erythrocytes and hemoglobin levels were increased by 10% in PTU-treated animals and higher increments (around 25%) were observed in these parameters when exogenous T(3) was coadministered. Dithiothreitol did not change blood delta-ALA-D activity of PTU-exposed animals when present in the reaction medium, suggesting no involvement of the enzyme's essential thiol groups in PTU-induced delta-ALA-D inhibition. PTU did not affect blood delta-ALA-D activity in vitro. These results are the first to show a correlation between hypothyroidism and decreased delta-ALA-D activity and point to this enzyme as a potential molecule involved with hypothyroidism-related hematological changes.     

罗伊乳酸杆菌甘油脱水酶基因的克隆及其在大肠杆菌中的表达

迅速升温的生物柴油投资热导致了其副产物甘油的大量积累,这一现状使得开发和利用甘油生产各种精细化工产品备受关注。本实验通过构建基因工程菌来生物转化甘油生产3-羟基丙醛,为甘油下游产品的开发开辟了一条新途径。3-羟基丙醛是一种重要的化学中间体,同时也是一种有效的抗菌剂和生物组织的固定剂,在化学工业中具有广泛的应用前景。实验主要利用甘油脱水酶N末端序列,并根据NCBI中公布的甘油脱水酶的氨基酸序列设计了一对克隆引物,并以菌株罗伊乳酸杆菌Lactobacillus reuteri的基因组DNA为模板进行PCR扩增,获得约为1.6kb的片段,将其克隆到T载体上进行测序,对测序结果进行分析,重新设计两端含有EcoRI和HindIII酶切位点的表达引物,利用PCR扩增得到了甘油脱水酶基因,该基因片段长度为1674bp,编码558个氨基酸。将所得片段定向克隆到pET28b载体中,并转化至大肠杆菌BL21感受态细胞中。经IPTG诱导后,进行SDS-PAGE电泳,在约65kD处检测出一蛋白表达条带,另外还对该重组菌进行比活力测定,最高比活力可达1.14U/mg,比野生型菌株提高了86.88%。