Graham J. Hart

Biosynthesis of the natural porphyrins: proof that hydroxymethylbilane synthase (porphobilinogen deaminase) uses a novel binding group in its catalytic action

Hydroxymethylbilane synthase builds a bilane by assembling 4 monopyrrolic units, the first of these being bound covalently to the enzyme through a group X; it is proved that X represents a unique enzymic cofactor based on a pyrromethane system.

13C-N.M.R. studies on the pyrromethane cofactor of hydroxymethylbilane synthase

By growing Escherichiacoli in the presence of 5-amino [5-13C] laevulinic acid, the enzyme hydroxymethylbilane synthase is produced carrying 13C-labels in its pyrromethane cofactor. It is then proved by 13C-n.m.r. spectroscopy that the cofactor is bound to the protein via the sulphur atom of a cysteine residue.

Biosynthesis of porphyrins and related macrocycles. Part 40. Synthesis of a spiro-lactam related to the proposed spiro-intermediate for porphyrin biosynthesis: inhibition of cosynthetase

Routes are developed for synthesis of the tripyrrolic macrocyclic spiro-lactam 39. A minor product from the synthesis, thought earlier to be an atropisomer, has been shown by molecular mechanics calculations and re-investigation to be a dimer.The octa-acid derived from 39 closely resembles the spiro-pyrrolenine 2 proposed as a biosynthetic intermediate for uroporphyrinogen Ill. This octa-acid acts as a strong inhibitor of cosynthetase (uroporphyrinogen Ill synthase) whilst other similar systems which lack some of its functionality do not. These results strongly support the view that the spiro system 2 is indeed the biosynthetic intermediate for formation of uroporphyrinogen III 3 from hydroxymethylbilane 1.

Biosynthesis of porphyrins and related macrocycles. Part 35. Discovery of a novel dipyrrolic cofactor essential for the catalytic action of hydroxymethylbilane synthase (porphobilinogen deaminase)

The enzyme hydroxymethylbilane synthase constructs the open-chain hydroxymethylbilane by assembly of four porphobilinogen units head-to-tail, the first of these being covalently bound to the enzyme through a group X. The surprising discovery is made that X is a novel dipyrromethane cofactor constructed from two porphobilinogen units and bound to the protein via the sulphur of cysteine. This cofactor does not turn over in the catalytic process but acts as an anchor for the assembly of a hexapyrrole from which the tetrapyrrolic hydroxymethylbilane is cleaved leaving the dipyrromethane cofactor in place for a further building cycle.

Stereochemistry of formation of the hydroxymethyl group of hydroxymethylbilane, the precursor of uro'gen-III

A new synthetic route to (11S)- and (11R)-[11-3H1]porphobilinogen (PBG) is described and the configurations of the products are assigned by degradation to (2S)- and (2R)-[2-3H1]glycollic acids; these PBG samples are used to establish that hydroxymethylbilane synthase converts PBG into the hydroxymethylbilane with overall retention of configuration at the HOCH2– group.

Biosynthesis of the pigments of life: Structure and mode of action of a novel enzymatic cofactor

Abstract Biosynthesis of the organic nuclei of hemes, chlorophylls, cytochromes, andvitamin B 12 involves, as a first stage, the building of a linear tetrapyrrole by the enzyme hydroxymethylbilane synthase. This enzyme has been found to use a novel dipyrrolic cofactor whose structure has been established. The function of this cofactor in the building process and the way in which the cofactor is bound to the enzyme have both been determined.

Synthesis of (11S)- and (11R)-[11-2H1]porphobilinogen; stereochemical studies on hydroxymethylbilane synthase (PBG deaminase)

(11S)-and (11R)-[11-2H1]Porphobilinogen (PBG) are synthesized and their configurations are established by degradation to a derivative of 2H1-glycine; they are used to prove that when hydroxymethylbilane synthase (PBG deaminase) acts on PBG in the presence of ammonia, aminomethylbilane is formed with overall retention of configuration at the NH2CHD-group.

Biosynthesis of porphyrins and related macrocycles. Part 52. Synthesis of (11-S)-[11-2H1]porphobilinogen and the (11R)-enantiomer for stereochemical studies on hydroxymethylbilane synthase (porphobilinogen deaminase)

A synthetic route is devised for the synthesis of (11S)-[11-2H1]porphobilinogen 1a and of the (11R)-enantiomer 1b. Their absolute configurations and enantiomeric purity are established by degradation to a derivative of [2-2H1]glycine of known stereochemistry. Methods are then developed, based on the synthesis of chiral imidate esters, for determination of the configuration of [2H1]-labelled aminomethylpyrroles by converting them into [2H1]-labelled amidines followed by analysis using 1H-NMR. The labelled samples of PBG 1a and 1b serve as substrates for hydroxymethylbilane synthase and the products are trapped as [2H1]-labelled aminomethylbilanes 7c and 7d. Their configurations are determined by the NMR assay to demonstrate that as PBG 1 is enzymically converted into the aminomethylbilane 7, there is overall retention of configuration at the aminomethyl carbon.

Biosynthesis of porphyrins and related macrocycles. Part 53. Stereochemical studies on the enzymic formation of hydroxymethylbilane, the precursor of uroporphyrinogen III

A new synthesis of porphobilinogen 1 (PBG) is described that allows the preparation of (11R)-[11-3H1]PBG 1a and its (11S)-enantiomer 1b. Their enantiomeric purities are determined by degradation of their immediate synthetic precursors by way of 3H-labelled glycines to yield two samples of 3H-labelled glycolic acid 16. The enzyme glycolate oxidase, known to remove HR stereospecifically from the methylene group of glycolic acid in forming glyoxylic acid 17, is then used to assay the configurations of these two samples. Each 3H-labelled PBG 1a and 1b is converted by hydroxymethylbilane synthase into hydroxymethylbilane 5a and 5b. Methods are devised for the isolation of this labile product from water and for its subsequent degradation to two further samples of glycolic acid. These are assayed enzymically to prove that there is overall retention of configuration as the aminomethyl carbon of PBG 1 enzymically affords the hydroxymethyl centre of the bilane 5. Thus, the two covalent bonds that are formed in this whole process must both involve reactions with retention of configuration or both with inversion. The significance of these results is discussed.