20-Methylsirohydrochlorin. A revised structure for a trimethylisobacteriochlorin intermediate (factor III) in the biosynthesis of vitamin B12

Mueller, Gerhard O. W.; Gneuss, K.D.; Kriemler, Hans-Peter; Scott, A. Ian; Irwin, A. J.

doi:10.1021/ja00507a041

Cited by 39 publications

(18 citation statements)

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“…Very careful and delicate experiments with strict oxygen exclusion allowed Battersby and co-workers to determine the structure of the true intermediate as 15,23-dihydrosirohydrochlorin (compound 3) (6). The third step along the cobalamin pathway is a C-20 methylation (10,16,51). The trimethylated system, until now purified only as the ester of the oxidized form (factor III) (compound 8), is very likely to be biosynthesized and further transformed in the dihydro state (compound 4) (2), and the entire sequence from urogen III to cobyrinic acid probably occurs without any change of original oxidation level (4,60).…”

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“…Hepta-, hexa-, and pentacarboxyporphyrin III and factor III were isolated as methyl esters from cultures of strain SC510 (51), and factors I and II were isolated from cultures of strain SC510 Rifr (pXL190) (16,51). Cobester dicyanide was obtained by methanolysis of vitamin B12 (7).…”

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“…Porphyrin esters were hydrolyzed and reduced to porphyrinogens as described previously (63). Methyl esters of factors I, II, and III as well as cobester were hydrolyzed in 2 M degassed piperidine (51). Factor I was reduced by the procedure described for porphyrins (42).…”

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“…Tetrapyrrolic acids were trapped onto a small (5 ml) DEAETrisacryl M column, esterified anaerobically with methanolsulfuric acid (95:5 [vol/vol]), and extracted as originally described (26). Methylated products were purified to constant specific activity by thin-layer chromatography as previously described (15,26,51 Fig. 4), for which the Prosolveur program was used (La Commande Electronique, Saint-Pierre De Bailleul, France).…”

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Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans

et al. 1989

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S-Adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT), the enzyme of the cobalamin biosynthetic pathway which catalyzes C methylation of uroporphyrinogen III, was purified about 150-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain by ammonium sulfate fractionation, anion-exchange chromatography, and hydroxyapatite chromatography. The purified protein has an isoelectric point of 6.4 and molecular weights of 56,500 as estimated by gel filtration and 30,000 as estimated by gel electrophoresis under denaturing conditions, suggesting that the active enzyme is a homodimer. It does not contain a chromophoric prosthetic group and does not seem to require metal ions or cofactors for activity. SUMT catalyzes the two successive C-2 and C-7 methylation reactions involved in the conversion of uroporphyrinogen III to precorrin-2 via the intermediate formation of precorrin-1. In vitro studies suggest that the intermediate monomethylated product (precorrin-1) is released from the protein and then added back to the enzyme for the second C-methylation reaction. The pH optimum was 7.7, the Km values for S-adenosyl-L-methionine and uroporphyrinogen III were 6.3 and 1.0 microM, respectively, and the turnover number was 38 h-1. The enzyme activity was shown to be completely insensitive to feedback inhibition by cobalamin and corrinoid intermediates tested at physiological concentration. At uroporphyrinogen III concentrations above 2 microM, SUMT exhibited a substrate inhibition phenomenon. It is suggested that this property might play a regulatory role in cobalamin biosynthesis in the cobalamin-overproducing strain studied.

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Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans

et al. 1989

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“…It was shown that SUMT is able to perform the first two methylation reactions at C-2 and C-7 on urogen III leading to precorrin-2 (compound 2). The following reaction along the cobalamin pathway is a methylation at C-20 on precorrin-2 that generates precorrin-3 (4,14). Precorrin-2 and precorrin-3 are highly oxygen-sensitive intermediates and are normally isolated in their aromatized forms as the esters sirohydrochlorin methyl ester (compound 6) and factor III methyl ester (compound 7).…”

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Assay and purification of S-adenosyl-L-methionine:precorrin-2 methyltransferase from Pseudomonas denitrificans

Thibaut¹,

Couder²,

Crouzet³

et al. 1990

J Bacteriol

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S-Adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), which catalyzes the C-20 methylation of precorrin-2 to precorrin-3, was purified to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans derived from a cobalamin-overproducing strain. Ammonium sulfate fractionation followed by chromatography on DEAE-Trisacryl, hydroxyapatite, and Mono Q HR purified the enzyme about 110-fold, with a 28% yield. For enzyme purification and characterization, a coupled-enzyme assay was developed which generated in situ the highly oxygen-sensitive substrate, precorrin-2, from 8-aminolevulinic acid. Evidence is given that the chemically reduced form of sirohydrochlorin (dihydrosirohydrochlorin) is methylated at C-20 to precorrin-3 by pure SP2MT. No subsequent SP2MT-dependent methylation reaction of precorrin-3 was detected. The native enzyme has an apparent molecular weight of 53,000, as estimated by gel filtration, and consists of two identical subunits of Mr 26,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Stepwise Edman degradation provided the N-terminal sequence of the first 17 amino acids.

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The Total Synthesis of Pyrrole Pigments 1973–1980

Jackson

1984

Total Synthesis of Natural Products

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20-Methylsirohydrochlorin. A revised structure for a trimethylisobacteriochlorin intermediate (factor III) in the biosynthesis of vitamin B12

Cited by 39 publications

References 1 publication

Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans

Purification and characterization of S-adenosyl-L-methionine: uroporphyrinogen III methyltransferase from Pseudomonas denitrificans

Assay and purification of S-adenosyl-L-methionine:precorrin-2 methyltransferase from Pseudomonas denitrificans

The Total Synthesis of Pyrrole Pigments 1973–1980

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