Cloning and sequence of the Salmonella typhimurium hemL gene and identification of the missing enzyme in hemL mutants as glutamate-1-semialdehyde aminotransferase.

Salmonella typhimurium forms the heme precursor delta-aminolevulinic acid (ALA) exclusively from glutamate via the five-carbon pathway, which also occurs in plants and some bacteria including Escherichia coli, rather than by ALA synthase-catalyzed condensation of glycine and succinyl-coenzyme A, which occurs in yeasts, fungi, animal cells, and some bacteria including Bradyrhizobium japonicum and Rhodobacter capsulatus. ALA-auxotrophic hemL mutant S. typhimurium cells are deficient in glutamate-1-semialdehyde (GSA) aminotransferase, the enzyme that catalyzes the last step of ALA synthesis via the five-carbon pathway. hemL cells transformed with a plasmid containing the S. typhimurium hemL gene did not require ALA for growth and had GSA aminotransferase activity. Growth in the presence of ALA did not appreciably affect the level of extractable GSA aminotransferase activity in wild-type cells or in hemL cells transformed with the hemL plasmid. These results indicate that GSA aminotransferase activity is required for in vivo ALA biosynthesis from glutamate. In contrast, extracts of both wild-type and hemL cells had gamma,delta-dioxovalerate aminotransferase activity, which indicates that this reaction is not catalyzed by GSA aminotransferase and that the enzyme is not encoded by the hemL gene. The S. typhimurium hemL gene was sequenced and determined to contain an open reading frame of 426 codons encoding a 45.3-kDa polypeptide. The sequence of the hemL gene bears no recognizable similarity to the hemA gene of S. typhimurium or E. coli, which encodes glutamyl-tRNA reductase, or to the hemA genes of B. japonicum or R. capsulatus, which encode ALA synthase. The predicted hemL gene product does show greater than 50% identity to barley GSA aminotransferase over its entire length. Sequence similarity to other aminotransferases was also detected.     

5-Aminolevulinic acid synthesis in Escherichia coli requires expression of hemA.

hemA and hemM, which are 213 bp apart and divergently transcribed, were separately cloned. We found that hemA is required for 5-aminolevulinic acid (ALA) synthesis in two ALA- auxotrophs. Overexpression of hemM alone did not produce ALA. More ALA was produced by strains harboring a plasmid with both hemA and hemM than by those with hemA alone. We conclude that hemA alone is required for ALA synthesis but hemA and hemM are required for maximal ALA synthesis.     

Cloning and structure of the hem A gene of Escherichia coli K-12

An Escherichia coli gene, which complements two independent hemA mutants of E. coli, has been cloned onto a multi-copy plasmid and both its strands have been sequenced. Both complemented mutants produce 5-aminolevulinic acid (ALA) and display fluorescence after 24h. The cloned sequence appears to encode a 46-kDa protein, which when produced in the maxicell procedure is processed to a 41-kDa protein as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. The amino acid sequence of the cloned gene product shows no significant homologies with any cloned ALA synthase, nor with any protein, in two E. coli databanks. A second cloned gene fragment, which has its coding region 34 bp away from the coding region of the gene that complements hemA, has been identified as part of protein release factor 1(RF1), thus confirming the location of hemA at min 26.7 and mapping it precisely near RF1. We have shown that E. coli utilizes the intact five-carbon chain of glutamate for the synthesis of ALA [Li et al., J Bacteriol. 171 (1989b) 2547-2552].     

Cloning and characterization of the gene encoding glutamate 1-semialdehyde 2,1-aminomutase, which is involved in delta-aminolevulinic acid synthesis in Propionibacterium freudenreichii.

The gene from Propionibacterium freudenreichii that encodes glutamate 1-semialdehyde 2,1-aminomutase (EC 5.4.3.8), which is involved in the C5 pathway for synthesis of delta-aminolevulinic acid (ALA), a precursor in heme and cobalamin biosynthesis, was cloned onto a multicopy plasmid, pUC18, via complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of fragments from the initial 3.3-kb chromosomal fragment allowed the isolation of a 1.9-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.9-kb DNA fragment revealed an open reading frame (ORF) that was located downstream from a potential ribosome-binding site. The ORF encoded a polypeptide of 441 amino acid residues, and the deduced molecular mass of this polypeptide is 45,932 Da. A high G+C content (70 mol%) of the codons of the ORF was found and was consistent with the taxonomic features of Propionibacterium species. The amino acid sequence showed a high degree of homology with those of the HemL proteins from other organisms, and a putative binding site for pyridoxal 5'-phosphate was conserved, with the exception of a single substitution of phenylalanine for leucine. These results suggest that ALA is synthesized via the C5 pathway in a producer of vitamin B12, P. freudenreichii.     

The Bacillus subtilis hemAXCDBL gene cluster, which encodes enzymes of the biosynthetic pathway from glutamate to uroporphyrinogen III.

We have recently reported (M. Petricek, L. Rutberg, I. Schr?der, and L. Hederstedt, J. Bacteriol. 172: 2250-2258, 1990) the cloning and sequence of a Bacillus subtilis chromosomal DNA fragment containing hemA proposed to encode the NAD(P)H-dependent glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid (ALA) synthesis, hemX encoding a hydrophobic protein of unknown function, and hemC encoding hydroxymethylbilane synthase. In the present communication, we report the sequences and identities of three additional hem genes located immediately downstreatm of hemC, namely, hemD encoding uroporphyrinogen III synthase, hemB encoding porphobilinogen synthase, and hemL encoding glutamate-1-semialdehyde 2,1-aminotransferase. The six genes are proposed to constitute a hem operon encoding enzymes required for the synthesis of uroporphyrinogen III from glutamyl-tRNA. hemA, hemB, hemC, and hemD have all been shown to be essential for heme synthesis. However, deletion of an internal 427-bp fragment of hemL did not create a growth requirement for ALA or heme, indicating that formation of ALA from glutamate-1-semialdehyde can occur spontaneously in vivo or that this reaction may also be catalyzed by other enzymes. An analysis of B. subtilis carrying integrated plasmids or deletions-substitutions in or downstream of hemL indicates that no further genes in heme synthesis are part of the proposed hem operon.     

Control of hemA expression in Rhodobacter sphaeroides 2.4.1: effect of a transposon insertion in the hbdA gene

The common precursor to all tetrapyrroles is 5-aminolevulinic acid (ALA), and in Rhodobacter sphaeroides its formation occurs via the Shemin pathway. ALA synthase activity is encoded by two differentially regulated genes in R. sphaeroides 2.4.1: hemA and hemT. In our investigations of hemA regulation, we applied transposon mutagenesis under aerobic conditions, followed by a selection that identified transposon insertion mutants in which hemA expression is elevated. One of these mutants has been characterized previously (J. Zeilstra-Ryalls and S. Kaplan, J. Bacteriol. 178:985-993, 1996), and here we describe our analysis of a second mutant strain. The transposon inserted into the coding sequences of hbdA, coding for S-(+)-beta-hydroxybutyryl-coenzyme A dehydrogenase and catalyzing an NAD-dependent reaction. We provide evidence that the hbdA gene product participates in polyhydroxybutyrate (PHB) metabolism and, based on our findings, we discuss possibilities as to how defective PHB metabolism might alter the level of hemA expression.     

5-aminolevulinic acid biosynthesis in Escherichia coli coexpressing NADP-dependent malic enzyme and 5-aminolevulinate synthase

5-aminolevulinate (ALA) synthase (E.C. 2.3.1.37), which mediates the pyridoxal phosphate-dependent condensation of glycine and succinyl-CoA, encoded by the Rhodobacter sphaeroides hemA gene, enables Escherichia coli strains to produce ALA at a low level. To study the effect of the enhanced C4 metabolism of E. coli on ALA biosynthesis, NADP-dependent malic enzyme (maeB, E.C. 1.1.1.40) was coexpressed with ALA synthase in E. coli. The concentration of ALA was two times greater in cells coexpressing maeB and hemA than in cells expressing hemA alone under anaerobic conditions with medium containing glucose and glycine. Enhanced ALA synthase activity via coupled expression of hemA and maeB may lead to metabolic engineering of E. coli capable of large-scale ALA production.     

5-Aminolevulinic acid synthesis in Escherichia coli.

A hemA mutant of Escherichia coli containing a multicopy plasmid which complemented the mutation excreted 5-aminolevulinic acid (ALA) into the medium. [1-14C]glutamate was substantially incorporated into ALA by this strain, whereas [2-14C]glycine was not. Periodate degradation of labeled ALA showed that C-5 of ALA was derived from C-1 of glutamate. The synthesis of ALA by two sonicate fractions which had been processed by gel filtration and dialysis, respectively, was dependent on glutamate, ATP, NADPH, tRNA(Glu), and pyridoxal phosphate. tRNA(Glu) stimulated ALA synthesis in a concentration-dependent manner. Pretreatment with RNase reduced this stimulation. The amino acid sequence of the cloned insert, derived from the nucleotide sequence (J.-M. Li, C. S. Russell, and S. D. Cosloy, J. Cell Biol. 107:617a, 1988), showed no homology with any ALA synthase sequenced to date. These results suggest that E. coli synthesizes ALA by the C5 pathway from the intact five-carbon chain of glutamate.     

Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium.

The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). Mutations in two genes, hemA and hemL, result in auxotrophy for ALA in Salmonella typhimurium, but the roles played by these genes and the mechanism of ALA synthesis are not understood. I have cloned and sequenced the S. typhimurium hemA gene. The predicted polypeptide sequence for the HemA protein shows no similarity to known ALA synthases, and no ALA synthase activity was detected in extracts prepared from strains carrying the cloned hemA gene. Genetic analysis, DNA sequencing of amber mutations, and maxicell studies proved that the open reading frame identified in the DNA sequence encodes HemA. Another surprising finding of this study is that hemA lies directly upstream of prfA, which encodes peptide chain release factor 1 (RF-1). A hemA::Kan insertion mutation, constructed in vitro, was transferred to the chromosome and used to show that these two genes form an operon. The hemA gene ends with an amber codon, recognized by RF-1. I suggest a model for autogenous control of prfA expression by translation reinitiation.     

Role of the hemA gene product and delta-aminolevulinic acid in regulation of Escherichia coli heme synthesis.

We initiated these studies to help clarify the roles of heme, delta-aminolevulinic acid (ALA), hemA, and hemM in Escherichia coli heme synthesis. Using recombinant human hemoglobin (rHb1.1) as a tool for increasing E. coli's heme requirements, we demonstrated that heme is a feedback inhibitor of heme synthesis. Cooverexpression of rHb1.1 and the hemA-encoded glutamyl-tRNA (GTR) reductase increased intracellular levels of ALA and heme and increased the rate of rHb1.1 formation. These results support the conclusion that heme synthesis is limited by ALA (S. Hino and A. Ishida, Enzyme 16:42-49, 1973; W. K. Philipp-Dormston and M. Doss, Enzyme 16:57-64, 1973) and that the hemA-encoded GTR reductase is a rate-limiting enzyme in the pathway (J.-M. Li, C. S. Russell, and S. D. Cosloy, Gene 82:2099-217, 1989). Increasing the copy number of hemM, whose product is believed to be required for efficient ALA formation (W. Chen, C. S. Russell, Y. Murooka, and S. D. Cosloy, J. Bacteriol. 176:2743-2746, 1994; M. Ikemi, K. Murakami, M. Hashimoto, and Y. Murooka, Gene 121:127-132, 1992), had no effect on either ALA pools or the rate of rHb1.1 accumulation. The hemA-encoded GTR reductase was found to be regulated by ALA. Some of our results differ from those reported by Hart and coworkers (R. A. Hart, P. T. Kallio, and J. E. Bailey, Appl. Environ. Microbiol. 60:2431-2437, 1994), who concluded that ALA formation is not the rate-limiting step in E. coli cells expressing Vitreoscilla hemoglobin.     

Cloning and sequencing of some genes responsible for porphyrin biosynthesis from the anaerobic bacterium Clostridium josui.

The 6.2-kbp DNA fragment encoding the enzymes in the porphyrin synthesis pathway of a cellulolytic anaerobe, Clostridium josui, was cloned into Escherichia coli and sequenced. This fragment contained four hem genes, hemA, hemC, hemD, and hemB, in order, which were homologous to the corresponding genes from E. coli and Bacillus subtilis. A typical promoter sequence was found only upstream of hemA, suggesting that these four genes were under the control of this promoter as an operon. The hemA and hemD genes cloned from C. josui were able to complement the hemA and hemD mutations, respectively, of E. coli. The COOH-terminal region of C. josui HemA and the NH2-terminal region of C. josui HemD were homologous to E. coli CysG (Met-1 to Leu-151) and to E. coli CysG (Asp-213 to Phe-454) and Pseudomonas denitrificans CobA, respectively. Furthermore, the cloned 6.2-kbp DNA fragment complemented E. coli cysG mutants. These results suggested that both C. josui hemA and hemD encode bifunctional enzymes.     

谷氨酸代谢调控及hemA过表达促进大肠杆菌血红素合成

【背景】大肠杆菌(Escherichia coli)以谷氨酸为前体经C5途径合成有限的血红素。【目的】探究胞内谷氨酸代谢及谷氨酰-tRNA还原酶基因(hem A)过表达对5-氨基乙酰丙酸(5-Aminolevulinic Acid,ALA)和血红素合成的影响。【方法】通过Red同源重组敲除与谷氨酸代谢有关的mscS与aroG,构建hemA表达载体并导入基因缺失菌株中。【结果】mscS单敲除或mscS与aroG双敲除对菌体生长无显著影响。与出发菌株相比,单敲除与双敲除菌株的谷氨酸含量均有所增加,ALA含量略微下降,血红素含量分别增加了11.6%和35.7%。在双敲除菌株中进一步过表达hemA后,胞内血红素含量增至47.603μmol/L。【结论】通过调控谷氨酸代谢流量与过表达hemA可促进血红素的合成,该结果为增强C5途径的血红素合成提供了新的思路。     

delta-Aminolevulinic acid dehydratase deficiency can cause delta-aminolevulinate auxotrophy in Escherichia coli

Ethylmethane sulfonate-induced mutants of several Escherichia coli strains that required delta-aminolevulinic acid (ALA) for growth were isolated by penicillin enrichment or by selection for respiratory-defective strains resistant to the aminoglycoside antibiotic kanamycin. Three classes of mutants were obtained. Two-thirds of the strains were mutants in hemA. Representative of a third of the mutations was the hem-201 mutation. This mutation was mapped to min 8.6 to 8.7. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding phage 8F10 allowed the isolation of the gene. DNA sequence analysis revealed that the hem-201 gene encoded ALA dehydratase and was similar to a known hemB gene of E. coli. Complementation studies of hem-201 and hemB1 mutant strains with various hem-201 gene subfragments showed that hem-201 and the previously reported hemB1 mutation are in the same gene and that no other gene is required to complement the hem-201 mutant. ALA-forming activity from glutamate could not be detected by in vitro or in vivo assays. Extracts of hem-201 cells had drastically reduced ALA dehydratase levels, while cells transformed with the plasmid-encoded wild-type gene possessed highly elevated enzyme levels. The ALA requirement for growth, the lack of any ALA-forming enzymatic activity, and greatly reduced ALA dehydratase activity of the hem-201 strain suggest that a diffusible product of an enzyme in the heme biosynthetic pathway after ALA formation is involved in positive regulation of ALA biosynthesis. In contrast to the hem-201 mutant, previously isolated hemB mutants were not ALA auxotrophs and had no detectable ALA dehydratase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Escherichia coli hemL gene encodes glutamate 1-semialdehyde aminotransferase.

delta-Aminolevulinic acid (ALA), the first committed precursor of porphyrin biosynthesis, is formed in Escherichia coli by the C5 pathway in a three-step, tRNA-dependent transformation from glutamate. The first two enzymes of this pathway, glutamyl-tRNA synthetase and Glu-tRNA reductase, are known in E. coli (J. Lapointe and D. Söll, J. Biol. Chem. 247:4966-4974, 1972; D. Jahn, U. Michelsen, and D. Söll, J. Biol. Chem. 266:2542-2548, 1991). Here we present the mapping and cloning of the gene for the third enzyme, glutamate 1-semialdehyde (GSA) aminotransferase, and an initial characterization of the purified enzyme. Ethylmethane sulfonate-induced mutants of E. coli AB354 which required ALA for growth were isolated by selection for respiration-defective strains resistant to the aminoglycoside antibiotic kanamycin. Two mutations were mapped to min 4 at a locus named hemL. Map positions and resulting phenotypes suggest that hemL may be identical with the earlier described porphyrin biosynthesis mutation popC. Complementation of the auxotrophic phenotype by wild-type DNA from the corresponding clone pLC4-43 of the Clarke-Carbon bank (L. Clarke and J. Carbon, Cell 9:91-99, 1976) allowed the isolation of the gene. Physical mapping showed that hemL mapped clockwise next to fhuB. The hemL gene product was overexpressed and purified to apparent homogeneity. The pure protein efficiently converted GSA to ALA. The reaction was stimulated by the addition of pyridoxal 5' -phosphate or pyridoxamine 5' -phosphate and inhibited by gabaculine or aminooxyacetic acid. The molecular mass of the purified GSA aminotransferase under denaturing conditions was 40,000 Da, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has apparent native molecular mass of approximately 80,000 Da, as determined by rate zonal sedimentation on glycerol gradients and molecular sieving through Superose 12, which indicates a homodimeric alpha2, structure of the protein.     

重组Rhodobacter sphaeroides hemA表达的调控

RhodobactersphaeroideshemA编码5氨基乙酰丙酸合酶(ALAS),催化磷酸吡哆醛依赖性琥珀酰CoA和甘氨酸缩合成ALA.将R.spaeroideshemA导入E.coli进行表达,当hemA具有与lac启动子相同的转录方向时,ALAS有活性.lac启动子与hemA之间的距离会影响ALAS在不同培养基上的表达.E.coli宿主菌对ALAS表达、ALA产量有显著影响,在实验所用6种菌株中,E.coliDH1是最佳宿主菌(P<0.05).ALAS表达还与碳源有关,琥珀酸为碳源时,重组ALAS活性最高(P<0.05),以乳酸为碳源时,ALAS活性很低.重组ALAS活性也受培养基pH值影响,pH6.5时,活性最高(P<0.05).     

Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.     

Effect of culture conditions on production of 5-aminolevulinic acid by recombinant Escherichia coli

Aminolevulinic acid (ALA) is formed by the enzyme ALA synthase (hemA gene). Then ALA is converted to Porphobilinogen (PBG) by the ALA dehydratase (hemB gene). For the overproduction of ALA, we used an Escherichia coli BL21(DE3) containing a hemA gene from Bradyrhzobium japonicum, which was created in our previous work. The effects of pH on the ALA synthase and ALA dehydratase were investigated. The ALA synthase and ALA dehydratase activities were dependent on the pH of the medium, with maximal activities occurring at pH 6.5 and 8.0 respectively. At pH 6.5, extracellular ALA reached 23 mM in a jar-fermenter. In addition, the effects of some nutritional factors, such as nitrogen source and the ratio of carbon to nitrogen (C/N) on the fermentative production of ALA were investigated. The highest ALA production was found with 8:1 of C/N ratio. Among various nitrogen sources, the tryptone might be a useful one for ALA production.     

The third member of the hemA gene family encoding glutamyl-tRNA reductase is primarily expressed in roots in Hordeum vulgare

Accumulation of chlorophylls and heme is primarily controlled at the level of 5-aminolevulinate (ALA) synthesis in higher plants. ALA is formed from glutamate in three enzymatic steps in plants. Among them, the reduction of glutamyl-tRNA^Gluto glutamate-1-semialdehyde (GSA) is likely to be a regulatory point of ALA synthesis. This reaction is catalyzed by glutamyl-tRNA reductase (GTR), which is encoded by a hemA gene. We have isolated a novel isoform of a hemA cDNA clone from barley (Hordeum vulgare) that is the third member of the hemA gene family. mRNA of this isoform is accumulated primarily in roots, suggesting that the isoform is regulated in an organ-specific manner by the demand for heme synthesis rather than chlorophyll. Phylogenetic analysis was done using the deduced amino acid sequences of hemA isoforms from barley, cucumber and Arabidopsis thaliana. The results indicate that the existing gene families in these plants arose after the divergence of monocotyledonous and dicotyledonous plants.