Overexpression in Escherichia coli of 12 Vitamin B12 Biosynthetic Enzymes

Roessner, Charles A.; Spencer, Jonathan B.; Ozaki, Shin‐ichi; Min, Changwook; Atshaves, Barbara P.; Nayar, P. G. N.; Anousis, Nick; Stolowich, Neal J.; Holderman, Marc T.; Scott, A. Ian

doi:10.1006/prep.1995.1019

Cited by 45 publications

(22 citation statements)

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“…Site-directed Mutagenesis of E. coli PBGS and Human PBGS-The plasmid pCR261, containing the E. coli hemB gene for PBGS, was a kind gift of Dr. C. Roessner of Texas A & M, College Station, TX (21). A 1400-base pair fragment containing the 1100-base pair hemB gene was excised from the EcoRI and BamHI site of pCR261 and inserted into the multiple cloning site of pUC119 to form pLM1228.…”

Section: Methodsmentioning

confidence: 99%

Mechanistic Implications of Mutations to the Active Site Lysine of Porphobilinogen Synthase

Mitchell¹,

Volin

Martins

et al. 2001

Journal of Biological Chemistry

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Porphobilinogen synthase (PBGS) is a homo-octameric protein that catalyzes the complex asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA). The only characterized intermediate in the PBGS-catalyzed reaction is a Schiff base that forms between the first ALA that binds and a conserved lysine, which in Escherichia coli PBGS is Lys-246 and in human PBGS is Lys-252. In this study, E. coli PBGS mutants K246H, K246M, K246W, K246N, and K246G and human PBGS mutant K252G were characterized. Alterations to this lysine result in a disabled but not totally inactive protein suggesting an alternate mechanism in which proximity and orientation are major catalytic devices. 13C NMR studies of [3,5-13 C]porphobilinogen bound at the active sites of the E. coli PBGS and the mutants show only minor chemical shift differences, i.e. environmental alterations. Mammalian PBGS is established to have four functional active sites, whereas the crystal structure of E. coli PBGS shows eight spatially distinct and structurally equivalent subunits. Biochemical data for E. coli PBGS have been interpreted to support both four and eight active sites. A unifying hypothesis is that formation of the Schiff base between this lysine and ALA triggers a conformational change that results in asymmetry. Product binding studies with wild-type E. coli PBGS and K246G demonstrate that both bind porphobilinogen at four per octamer although the latter cannot form the Schiff base from substrate. Thus, formation of the lysine to ALA Schiff base is not required to initiate the asymmetry that results in half-site reactivity.Porphobilinogen synthase (PBGS, 1 also known as 5-aminolevulinate dehydratase) is a metalloenzyme that catalyzes the asymmetric condensation of two molecules of 5-aminolevulinic acid (ALA) to form porphobilinogen, the monopyrrole precursor of tetrapyrroles (see Fig. 1). Although PBGS proteins from different organisms differ in their use of metal ions, there is remarkable sequence conservation between the PBGS from eubacteria, archaea, and eucaryotes implying a commonality in the overall protein architecture and reaction mechanism (1). The two PBGS studied herein, those of Escherichia coli and human, both require a catalytic Zn(II), neither require monovalent cations, and the E. coli PBGS activity is stimulated by an allosteric Mg(II) (2-5). The overall protein sequence identity between E. coli and human PBGS is 42%, and the active site residues are significantly more conserved.Considerable effort has been applied to the characterization of PBGS proteins from various organisms, and several crystal structures are now available (6 -10). Yet much of the chemical reaction mechanism remains speculative (7). Only one intermediate, an enzyme-substrate Schiff base, has been identified (11). Several x-ray crystal structures contain levulinic acid bound in a fashion analogous to the Schiff base (Protein Database codes 1B4K, 1YLV, and 1B4E), and the actual Schiff base involving ALA has been observed by 13 C and 15 N NMR for a chemically mo...

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Section: Methodsmentioning

confidence: 99%

Mechanistic Implications of Mutations to the Active Site Lysine of Porphobilinogen Synthase

Mitchell¹,

Volin

Martins

et al. 2001

Journal of Biological Chemistry

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“…All other chemicals were purchased from Sigma Chemical Co. and were of the highest grade obtainable. ALA dehydratase, porphobilinogen deaminase, urogen III synthase (cosynthetase), CysG, and precorrin-2 methyltransferase were isolated from recombinant strains of E. coli as previously described (22,26,27,34). Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs or Boehringer Mannheim and used as directed by the supplier.…”

Section: Methodsmentioning

confidence: 99%

“…Urogen I and urogen III were synthesized from ALA in a multienzyme reaction with enzymes isolated from recombinant strains of E. coli as previously described (16,26). Briefly, ALA (3 to 5 mg) was first converted to porphobilinogen in a reaction mixture containing 100 ml of 0.1 M Tris-HCl, pH 8.0, and 15 mg of ALA dehydratase.…”

Section: Methodsmentioning

confidence: 99%

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii)

et al. 1995

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We cloned, sequenced, and overexpressed cobA, the gene encoding uroporphyrinogen III methyltransferase in Propionibacterium freudenreichii, and examined the catalytic properties of the enzyme. The methyltransferase is similar in mass (27 kDa) and homologous to the one isolated from Pseudomonas denitrificans. In contrast to the much larger isoenzyme encoded by the cysG gene of Escherichia coli (52 kDa), the P. freudenreichii enzyme does not contain the additional 22-kDa peptide moiety at its N-terminal end bearing the oxidase-ferrochelatase activity responsible for the conversion of dihydrosirohydrochlorin (precorrin-2) to siroheme. Since it does not contain this moiety, it is not a likely candidate for synthesis of a cobalt-containing early intermediate that has been proposed for the vitamin B 12 biosynthetic pathway in P. freudenreichii. Uroporphyrinogen III methyltransferase of P. freudenreichii not only catalyzes the addition of two methyl groups to uroporphyrinogen III to afford the early vitamin B 12 intermediate, precorrin-2, but also has an overmethylation property that catalyzes the synthesis of several tri-and tetra-methylated compounds that are not part of the vitamin B 12 pathway. The enzyme catalyzes the addition of three methyl groups to uroporphyrinogen I to form trimethylpyrrocorphin, the intermediate necessary for biosynthesis of the natural products, factors S1 and S3, previously isolated from this organism. A second gene found upstream from the cobA gene encodes a protein homologous to CbiO of Salmonella typhimurium, a membrane-bound, ATP-dependent transport protein thought to be part of the cobalt transport system involved in vitamin B 12 synthesis. These two genes do not appear to constitute part of an extensive cobalamin operon.Uroporphyrinogen (urogen) III methyltransferase, a key enzyme in the biosynthetic pathways of vitamin B 12 and siroheme, catalyzes the S-adenosyl-L-methionine (SAM)-dependent bismethylation of its substrate, urogen III, resulting in the formation of dihydrosirohydrochlorin (known as precorrin-2 in the vitamin B 12 pathway). In the biosynthesis of vitamin B 12 in the anaerobe Propionibacterium freudenreichii, labeling experiments have indicated that cobalt is inserted soon after the formation of precorrin-2 (3, 20), and the recent isolation of a cobalt-containing tetramethylated corphinoid, possibly an intermediate, from this organism supports these observations (3). Cobalt insertion in the vitamin B 12 pathway of the aerobe Pseudomonas denitrificans, however, occurs at a much later stage with insertion into hydrogenobyrinic acid diamide (6).Urogen III methyltransferase has been purified to homogeneity from several different organisms, and the nucleotide sequences of the corresponding genes have been determined, revealing that the enzyme exists in at least two forms. One form, encoded by the cysG gene, is required for siroheme and, thus, for cysteine synthesis in Escherichia coli (14,31,35) and siroheme and vitamin B 12 synthesis in Salmonella typhimurium (8, 10). The ...

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“…For a novel approach to heterologous expression of human PBGS, we chose to mimic a construct used for overexpression of the E. coli hemB gene in E. coli (14,15). This system can generate hundreds of milligrams of E. coli PBGS (up to 30% of the soluble protein).…”

mentioning

confidence: 99%

An Artificial Gene for Human Porphobilinogen Synthase Allows Comparison of an Allelic Variation Implicated in Susceptibility to Lead Poisoning

Jaffe

Volin

Bronson-Mullins

et al. 2000

Journal of Biological Chemistry

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Porphobilinogen synthase (PBGS) is an ancient enzyme essential to tetrapyrrole biosynthesis (e.g. heme, chlorophyll, and vitamin B 12 ). Two common alleles encoding human PBGS, K59 and N59, have been correlated with differential susceptibility of humans to lead poisoning. However, a model for human PBGS based on homologous crystal structures shows the location of the allelic variation to be distant from the active site with its two Zn(II). Previous microbial expression systems for human PBGS have resulted in a poor yield. Here, an artificial gene encoding human PBGS was constructed by recursive polymerase chain reaction from synthetic oligonucleotides to rectify this problem. The artificial gene was made to resemble the highly expressed homologous Escherichia coli hemB gene and to remove rare codons that can confound heterologous protein expression in E. coli. We have expressed and purified recombinant human PBGS variants K59 and N59 in 100-mg quantities. Both human PBGS proteins purified with eight Zn(II)/octamer; Zn(II) binding was shown to be pH-dependent; and Pb(II) could displace some of the Zn(II). However, there was no differential displacement of Zn(II) by Pb(II) between K59 and N59, and simple Pb(II) inhibition studies revealed no allelic difference.

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Overexpression in Escherichia coli of 12 Vitamin B12 Biosynthetic Enzymes

Cited by 45 publications

References 0 publications

Mechanistic Implications of Mutations to the Active Site Lysine of Porphobilinogen Synthase

Mechanistic Implications of Mutations to the Active Site Lysine of Porphobilinogen Synthase

Cloning, sequencing, and expression of the uroporphyrinogen III methyltransferase cobA gene of Propionibacterium freudenreichii (shermanii)

An Artificial Gene for Human Porphobilinogen Synthase Allows Comparison of an Allelic Variation Implicated in Susceptibility to Lead Poisoning

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