Enzymatic mechanisms in biotin synthesis : vitamin B6 catalysis and phosphoryl transfer

Abstract

With the large number of pathogenic bacteria that have developed antibiotic resistance it is increasingly important to find new targets for antibiotics. Our interest in the biotin biosynthesis pathway originates from this drug design perspective. All organisms require biotin, but it is only synthesised in bacteria, in plants and in some fungi.

Mammals obtain biotin from the diet and from intestinal bacteria. The fact that the enzymes involved in synthesising biotin are present in bacteria and in plants, but do not exist in humans make them promising targets for new antibiotics and herbicides. In Order to obtain information valuable for drug design, we have studied the active sites of two of the enzymes involved in biotin production in Escherichia coli in detail.

Dethiobiotin synthetase catalyses the third step of biotin biosynthesis, the closure of the ureido ring of biotin. The reaction requires ATP and magnesium. We have cocrystallised this enzyme with ADP and aluminium fluoride, in an attempt to obtain a mimic of the transition state of the phosphoryl transfer step, where a phosphate group is transferred from ATP to the substrate. The crystal structure of the complex revealed an aluminium fluoride molecule bound as A1F3 in a tetrahedral conformation, mimicking the gamma-phosphate after phosphoryl transfer has occurred rather than the planar transition state.

7,8-diaminopelargonic acid synthase is a pyridoxal-5'-phosphate-dependent aminotransferase that catalyses the second step of the pathway. It is unique in that it utilises S-adenosyl-methionine as an amino group donor. On the basis of the crystal structure of a non-productive complex between the enzyme and the amino acceptor substrate, 7-keto-8-aminopelargonic acid, we have identified a number of residues that are involved in substrate binding. These amino acids have been systematically replaced by site directed mutagenesis. The resulting mutant proteins have been crystallised and their three-dimensional structure have been determined. A selection of these mutants have been kinetically characterised.

The results of the study suggests that the two substrates bind in nearly completely distinct sites in the vicinity of the cofactor. Furthermore, we have determined the crystal structure of 7,8-diaminopelargonic acid synthase in complex with amiclenomycin, a naturally occurring antibiotic. The structure revealed that the molecule adopts the same position as 7-keto-8-aminopelargonic acid and forms a covalent linkage with the cofactor. This information is valuable for the design of novel antibiotics.