5-Aminolevulinic acid synthesis in Escherichia coli

Li, Jianming; Brathwaite, Ormond; Cosloy, Sharon D.; Russell, C.S.

doi:10.1128/jb.171.5.2547-2552.1989

Cited by 91 publications

(63 citation statements)

References 44 publications

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“…The use of the E. coli hemA Ϫ HU227 strain (32) as the initial biological screen, although powerful, had some limitations. Specifically, the inability of providing heme prototrophy to this strain could be due to inappropriate folding, proteolytic degradation, malfunction in PLP binding, or assembly of the two subunits or could just emulate ALAS variants, which had enzymatic activities too low to compensate the ALA and heme requirements of the hemA Ϫ strain (32).…”

Section: Steady-state Kinetic Characterization Of Circularly Permutedmentioning

confidence: 99%

“…Specifically, the inability of providing heme prototrophy to this strain could be due to inappropriate folding, proteolytic degradation, malfunction in PLP binding, or assembly of the two subunits or could just emulate ALAS variants, which had enzymatic activities too low to compensate the ALA and heme requirements of the hemA Ϫ strain (32). By studying the active, circularly permuted ALAS variants (Fig.…”

Section: Steady-state Kinetic Characterization Of Circularly Permutedmentioning

confidence: 99%

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Circular Permutation of 5-Aminolevulinate Synthase

Cheltsov¹,

Barber²,

Ferreira

2001

Journal of Biological Chemistry

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5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in non-plant eukaryotes and some prokaryotes. The enzyme functions as a homodimer and requires pyridoxal 5-phosphate as a cofactor. Although the roles of defined amino acids in the active site and catalytic mechanism have been recently explored using site-directed mutagenesis, much less is known about the role of the 5-aminolevulinate synthase polypeptide chain arrangement in folding, structure, and ultimately, function. To assess the importance of the continuity of the polypeptide chain, circularly permuted 5-aminolevulinate synthase variants were constructed through either rational design or screening of an engineered random library. One percent of the random library clones were active, and a total of 21 active variants had sequences different from that of the wild type 5-aminolevulinate synthase. Out of these 21 variants, 9 displayed unique circular permutations of the 5-aminolevulinate synthase polypeptide chain. The new termini of the active variants disrupted secondary structure elements and loop regions and fell in 100 amino acid regions from each terminus. This indicates that the natural continuity of the 5-aminolevulinate synthase polypeptide chain and the sequential arrangement of the secondary structure elements are not requirements for proper folding, binding of the cofactor, or assembly of the two subunits. Furthermore, the order of two identified functional elements (i.e. the catalytic and the glycine-binding domains) is apparently irrelevant for proper functioning of the enzyme. Although the wild type 5-aminolevulinate synthase and the circularly permuted variants appear to have similar, predicted overall tertiary structures, they exhibit differences in the arrangement of the secondary structure elements and in the cofactor-binding site environment. Taken together, the data lead us to propose that the 5-aminolevulinate synthase overall structure can be reached through multiple or alternative folding pathways.

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Section: Steady-state Kinetic Characterization Of Circularly Permutedmentioning

confidence: 99%

Section: Steady-state Kinetic Characterization Of Circularly Permutedmentioning

confidence: 99%

Circular Permutation of 5-Aminolevulinate Synthase

Cheltsov¹,

Barber²,

Ferreira

2001

Journal of Biological Chemistry

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“…In the present work, neither soybean leaf extract nor nodule extract was able to catalyze the incorporation of radiolabel from l-['4C]glutamate into ALA, although labeled ALA was synthesized from 3,4-t3H]-glutamate ( Table I). Extracts of E. coli, which contain the C5 pathway (Avissar and Beale, 1989;Grimm et al, 1989;Li et al, 1989;Ilag et al, 1991), catalyzed the formation of radiolabeled ALA from either glutamate isotope ( Table I). The results show that the inability of the I-carbon of glutamate to be incorporated into ALA in soybean is not specific to root nodules because it was also observed in extracts of greening etiolated leaves, where the C5 pathway is presumed to operate and have maximal activity.…”

Section: Ala Formation From Clutamate and Gsamentioning

confidence: 99%

Expression of the Soybean (Glycine max) Glutamate 1-Semialdehyde Aminotransferase Gene in Symbiotic Root Nodules

Sangwan

O’Brian

1993

Plant Physiol.

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“…There are two distinct routes for the synthesis of this non-protein amino acid. In one route, used by animals, yeast, and bacteria such as Rhodobacter and Rhizobium, a single enzyme condenses glycine with succinyl-CoA to produce 6- (3). Additionally, the conversion of glutamate to 6-aminolevulinate in extracts of the hemA mutant was restored in vitro by the addition of crude preparations of the reductase from Chlorella (4).…”

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confidence: 95%

Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

Vothknecht

Kannangara

Wettstein

1996

Proc. Natl. Acad. Sci. U.S.A.

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ABSTRACT8-Aminolevulinate in plants, algae, cyanobacteria, and several other bacteria such as Escherichia coli and Bacillus subtilis is synthesized from glutamate by means of a tRNAGIU mediated pathway. The enzyme glutamyl tRNAGIU reductase catalyzes the second step in this pathway, the reduction of tRNA bound glutamate to give glutamate 1-semialdehyde. The hemA gene from barley encoding the glutamyl tRNAGIU reductase was expressed in E. coli cells joined at its amino terminal end to Schistosoma japonicum glutathione Stransferase (GST). GST-glutamyl tRNAGlU reductase fusion protein and the reductase released from it by thrombin digestion catalyzed the reduction of glutamyl tRNAGIlu to glutamate 1-semialdehyde. The specific activity of the fusion protein was 120 pmol ,ug-l min-1. The fusion protein used tRNAGIU from barley chloroplasts preferentially to E. coli tRNAGIU and its activity was inhibited by hemin. It migrated as an 82-kDa polypeptide with SDS/PAGE and eluted with an apparent molecular mass of 450 kDa from Superose 12. After removal of the GST by thrombin, the protein migrated as an -60-kDa polypeptide with SDS/PAGE, whereas gel filtration on Superose 12 yielded an apparent molecule mass of 250 kDa. Isolated fusion protein contained heme, which could be reduced by NADPH and oxidized by air.

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5-Aminolevulinic acid synthesis in Escherichia coli

Cited by 91 publications

References 44 publications

Circular Permutation of 5-Aminolevulinate Synthase

Circular Permutation of 5-Aminolevulinate Synthase

Expression of the Soybean (Glycine max) Glutamate 1-Semialdehyde Aminotransferase Gene in Symbiotic Root Nodules

Expression of catalytically active barley glutamyl tRNAGlu reductase in Escherichia coli as a fusion protein with glutathione S-transferase.

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