Alan R. Battersby

Studies of enzyme-mediated reactions. Part 12. Stereochemical course of the decarboxylation of (2S)-tyrosine to tyramine by microbial, mammalian, and plant systems.

Tyrosine is proved to be decarboxylated with retention of configuration by three aromatic amino-acid decarboxylases of bacterial, mammalian, and plant origin by experiments based on the synthesis of (αS)-[α-3H]tyrosine and the (αR)- and (αS)-isomers of [α-3H1]tyramine. Incubation of the labelled tyramines with the diamine oxidase from pea seedlings and the monoamine oxidase from rat liver shows that the former enzyme removes Hsi and the latter HRe. These amine oxidases are used to assay the chirality of the [α-3H1]tyramines produced by incubation of suitably labelled tyrosines in tritiated water or in unlabelled water with the partially purified decarboxylases from Streptococcus faecalis and hog kidney. The plant enzyme was studied by administration of (αS)-[α-3H,U-14C]tyrosine to intact Papaver somniferum plants followed by isolation of the resultant papaverine, which retained no tritium; morphine was also isolated to act as an internal standard.

Biosynthesis of Vitamin B12

Vitamin B12 (cyanocobalamin) is the normal isolated form of coenzyme B12 (adenosylcobalamin), a structure of marvellous architecture and amazing biological activity. It belongs to the family of tetrapyrroles which includes inter alia the haems and the chlorophylls. This review begins with a brief overview of the biosynthesis of tetrapyrroles in general but then concentrates on recent research on B12 biosynthesis. The first main section reviews the biosynthesis of uro’gen III, the last common precursor of all natural tetrapyrroles, concentrating particularly on the three enzymes, porphobilinogen synthase, hydroxymethylbilane synthase and uro’gen III synthase. Crystal structures are available for the second of these enzymes and a new proposal is presented for its detailed mode of action. The second main section reviews the recent discovery of the complete biosynthetic pathway from uro’gen III to hydrogen-obyrinic acid in Pseudomonas denitrificans, which has revealed some beautiful and totally unexpected chemistry. A short section then describes the many similarities and some differences in the chemistry used by the micro-aerophilic organism Propionibacterium shermanii for the synthesis of coenzyme B12 compared with that seen in the aerobic Ps. denitrificans. Finally an account is given of the remarkable steps needed to complete the synthesis of the coenzyme in both organisms.

Biosynthesis of porphyrins and related macrocycles : Part 27. Syntheses of modified hydroxymethylbilanes and studies of their chemical and biological properties

Abstract The biosynthetic pathway to uroporphyrinogen-III, the parent macrocycle for all the pigments of life, involves the formation and ring-closure of an hydroxymethylbilane. The non-enzymic ring-closure of this bilane is studied under different pH conditions. Also octamethyl esters of related bilanes are synthesised which have either a cyano or a methyl group blocking position-19 which is free on the terminal ring-D of the natural bilane. Studies are made of the ring-closure of these substituted bilanes under acidic conditions. The conclusion is reached that there is a strong preference for non-enzymic ring-closure of an hydroxymethyebilane to occur at the terminal carbon atom (position-19). The octa-acids derived from the cyano and methyl substituted bilanes inhibit the action of cosynthetase on the natural hydroxymethylbilane.

Studies of enzyme-mediated reactions. Part 13. Stereochemical course of the formation of histamine by decarboxylation of (2S)-histidine with enzymes from Clostridium welchii and Lactobacillus 30a

(αS)-[α-3H]Histidine is prepared and the [α-3H1]histamine produced from it by the decarboxylating enzymes of Clostridium welchii and Lactobacillus 30a is proved by assay with diamine oxidase from pea seedlings to have the (αS)-configuration. The (Si)-stereospecificity of the latter enzyme is confirmed for the case of histamine by studying its action on (αR)-[α-3H1]histamine (21) which is synthesised rationally. The combined results prove that histidine decarboxylases from both sources bring about decarboxylation with overall retention of configuration. It is shown that histidine decarboxylase acts on histamine to bring about slow exchange with the medium of the α-Re-hydrogen, which occupies the same space (Re-space) as the carboxy-group of (αS)-histidine. Syntheses are described of (αS,βS)-[β-3H1]histidine (45) and (αS,βR)-[β-3H1]histidine (47).

Uroporphyrinogen III synthase: Studies on its mechanism of action, molecular biology and biochemistry

Abstract A review is given of studies on the mechanism of action of uroporphyrinogen III synthase (cosynthetase, EC 4.2.1.75) based on synthesis of an inhibitory spiro-lactam. HemD, the Escherichia coli gene coding for uroporphyrinogen III synthase has been cloned and overexpressed at levels sixteen fold higher than in wild type E. coli. Evidence for hemD being part of an operon is reviewed. Uroporphyrinogen III synthase, which has been purified approx. 6000 fold, shows Mr 28000 under denaturing conditions and has a pH optimum of 8.0 ± 0.2. The results from chemical modification of the enzyme point to the presence of arginine and lysine residues at or close to the active site.

Synthetic studies relevant to biosynthetic research on vitamin B12. Part 7. Synthesis of (±)-bonellin dimethyl ester

The dimethyl ester of the green marine pigment, bonellin, has been synthesized in racemic form by a rational photochemical route. The two propionate residues of this biologically active pigment have been introduced synthetically in differentiated form which opens the way to the synthesis of the naturally occurring amino acid derivatives of bonellin.

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.

Biosynthesis of porphyrins and related macrocycles. Part III. Rational syntheses of [β-13C]-, [γ-13C]-, and [δ-13C]-protoporphyrin-IX: assignment of the 13C nuclear magnetic resonance signals from the meso-carbon atoms of protoporphyrin-IX dimethyl ester

The 13C signals arising from the meso-carbon atoms ot protoporphyrin-IX are of key importance for biosynthetic studies. Rigorous assignment is made of the signals from the β- and δ-carbon atoms by synthesis of [β-13C]- and [δ-13C]-protoporphyrin-IX dimethyl ester from unsymmetrical pyrromethenes in 42% yield. The formation of porphyrins from a,c-biladienes and formaldehyde is studied and leads to synthesis of [γ-13C]protoporphyrin-IX dimethyl ester. The spectrum of this product allows assignment of the two remaining signals, from the γ- and α-carbon atoms. The three 13C-labelled samples of protoporphyrin-IX dimethyl ester are converted into 2,4-diacetyldeuteroporphyrin-IX dimethyl ester; assignment is thereby made of the well-spread 13C signals from the meso-carbon atoms of this porphyrin. 13C Chemical shifts are reported for a variety of pyrrole derivatives and porphyrins.

Total synthesis of (±)-bonellin dimethyl ester

The dimethyl ester of the physiologically active green pigment bonellin from Bonellia viridis has been synthesised in racemic form by a sequence in which the macrocycle is formed by a photochemical ring-closure.

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.