No abstract

The organic nuclei of chlorophylls, haems, cytochromes and vitamin B12 are biosynthesised from a single tetrapyrrolic intermediate which has an unexpected, rearranged structure. The mechanism of biosynthesis of this key intermediate has now been characterised in detail. Some of the information thereby obtained is also of use in the investigation of human diseases such as the porphyrias.

Vitamin B,, is an essential vitamin for human health, and lack of it leads to pernicious anemia. This biological activity has attracted intense interest for some time; in addition, the complex architecture of the B,, molecule has fascinated chemists and biochemists since its discovery as the first natural organocobalt complex and the establishment of its structure by X-ray analysis. The organic ligand surrounding the cobalt displays many stereogenic centers along its periphery carrying reactive functional groups. This complexity led vitamin B,, to be rightly regarded as an extreme challenge to the synthetic chemist. Yet microorganisms achieve this synthesis in vivo with complete control of regio-and stereochemistry. How do they do it? This review tells the full remarkable story. Success in unraveling this biosynthetic puzzle resulted from a collaborative effort by biologists and chemists using the full range of methods available from their disciplines-from genetics at one end of the spectrum to synthesis and NMR spectroscopy at the other. This work can act as a guide for future research on the biosynthesis of yet more complex natural substances.

In part because humans cannot synthesize vitamin B12 and must obtain it from organisms that produce it and because B12 deficiency leads to pernicious anemia, it has been important to understand how microorganisms build this quite complex substance. As shown here, an interdisciplinary attack was needed, which combined the strengths of genetics, molecular biology, enzymology, chemistry, and spectroscopy. This allowed the step-by-step synthetic pathway of B12 to be elucidated, and this approach has acted as a model for future research on the synthesis of substances in living organisms. One practical outcome of such an approach has been the improved availability of B12 for animal feedstuffs and human health.

When the enzyme deaminase acts alone on porphobilinogen, it releases a transient intermediate into the medium which is unaffected by further treatment with a large excess of deaminase. The intermediate undergoes rapid ringclosure chemically (4 ca. 4 min) to form uraporphyrinogen-I. '3C Spectroscopic studies on the intermediate generated from 13C labelled porphobilinogen combined with synthesis of labelled standards for determination of chemical shifts establish its structure to be a linear tetrapyrrole, the unrearranged hydroxymethylbilane. Other workers deduced a different, cyclic structure (preuro'gen) which is shown here to be incorrect by chemical studies, l3C spectroscopy and 13C :15N double-labelling experiments. That the intermediate is the unrearranged hydroxymethylbilane is confirmed by its unambiguous synthesis. The natural and synthetic samples of this bilane are shown to be excellent and identical substrates for cosynthetase (free from deaminase) with production of uroporphyrinogen-Ill. Thus, deaminase is the enzyme for assembly of four porphobilinogen units to the linear tetrapyrrole stage and cosynthetase is the ring-closing and rearranging enzyme. Two proposals are discussed for the mechanism of inversion of the terminal ring-D of the hydroxymethylbilane in the formation of uroporphyrinogen- Ill.THE formation of uroporphyrinogen-111 (uro'gen-111) (3) and ammonia from four molecules of porphobilinogen (PBG) (1) is catalysed in living systems by two proteins, deaminase and cosynthetase. Cosynthetase is easily destroyed by heat-treatment and deaminase alone acts on PBG (1) to produce uroporphyrinogen-I (uro'gen-I) * This proposal grew as a result of a valuable discussion with Dr. D. C. Williams (Trinity College, Dublin) who we warmly thank, on the possible involvement of methylene-tetrahydrofolate in porphyrin biosynthesis.