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Researchers have been able to use an affinity chromatography resin as the basis for an efficient method for the purification of Escherichia coli porphobilinogen deaminase (PBG-D) apoenzyme. It was possible to identify circular dichroism (CD) spectra of apoenzyme and holoenzyme and marked differences in the backbone and aromatic region of the spectra. It was found that the reconstitution of apo- to holoenzyme took place much more quickly and more efficiently when preuroporphyrinogen was used as a substrate rather than PBG.

Electrospray mass spectrometric analysis of the influence of site-directed mutagenesis on two interdomain cysteine substrates at position 99 and 134 in Escherichi coli porphobilinogen deaminase shows that serine can replace cysteine as the dipyrromethane cofactor attachment site. X-ray structure of the deaminase indicates that a flexible hinge region acts as a medium for the interaction of the three enzyme domains. Progressive change in the position of domain 3 with respect to alpha-helix containing cysteine-134 is necessary for the exposure of cysteine-134.

A multiple-enzyme system containing 5-aminolevulinic acis (ALA) dehydratase, porphobilinogen deaminase, uro'gen II synthetase, s-adenosyl-L-metionine- (SAM)-dependent uro'gen III methyltransferase and precorrin-2 methyltransferase in the presence of (13C)SAM was used to produce the trimethylated intermediate in vitamin B12 (corrin) biosynthesis, precorrin. Precorrin closely resembles the structural conformation of precorrin-2, but with differences in the C=C and C=N bonds. The implications of this structure in corrin biosynthesis is discussed.

The studies about the two putative cobaltochelatase genomes, DVU0650 and DVU1365 from Desulfovibrio vulgaris Hildenborough, that share a significant degree of amino acid sequence similar to Salmonella typhimurium CbiK is reported. The cloning and characterization of the isolated enzymes provided insight into the role of these proteins and their involvement in modified tetrapyrrole biosynthesis in a Desulfovibrio species.

The enzyme biotin synthase (BioB), a member of the 'radical SAM' family, and equipped with iron-sulfur clusters and six conserved cysteins, catalyzes the final step of biotin biosynthesis. Mutants of a highly conserved region in BioB, the 'Tyr-150/Asn-151/His-152/Asn-153/Leu-154/Asp-155' sequence or the YNHNLD conserved motif, having properties similar to those of the wild-type enzyme in cluster content and characteristics, are characterized and their activities determined.

Research was conducted to examine the structure of the anaerobic cobalamin biosynthetic cobalt chelatase, CbiK, from Salmonella typhimurium to prove that it has a remarkably similar topology to Bacillus subtilis ferrochelatase despite minimal amino acid conservation. Several major residues within CbiK were mutated to investigate their involvement in the catalytic mechanism. The structural similarity has implications for the understanding of the evolution of this branched biosynthetic pathway.

A study was conducted to analyze the high resolution structure of a 5-aminolevulinic acid dehydratase (ALAD) from Escherichia coli complexed with levulinic acid. Results indicated that the carboxyl group of the levulinic acid moiety supports the formation of hydrogen bonds with the side chains of Ser 273 and Tyr 312. Findings also showed that a zinc ion supports the putative oxyanion intermediates in the reaction mechanism.