MECHANISM OF AN ATP-DEPENDENT CARBOXYLASE, DETHIOBIOTIN SYNTHETASE, BASED ON CRYSTALLOGRAPHIC STUDIES OF COMPLEXES WITH SUBSTRATES AND A REACTION INTERMEDIATE

The crystal structures of six complexes of homodimeric Escherichia col i dethiobiotin synthetase with a variety of substrates, substrate anal ogs, and products have been determined to high resolution. These inclu de (1) the binary complex of dethiobiotin synthetase and the N7-carbam ate of 7,8-diaminononanoic acid, (2) the binary complex of enzyme and the alternate substrate, 3-(1-aminoethyl)-nonanedioic acid, (3) the bi nary complex of enzyme with the product ADP, (4) the quaternary comple x of enzyme, ADP, the N7-carbamate of 7,8-diaminononanoic acid, and Ca 2+, (5) the ternary complex of enzyme, the ATP analog adenylyl (beta,g amma-methylene)diphosphonate, and the N7-carbamate of 7,8-diaminononan oic acid, and (6) the quaternary complex of enzyme, the ATP analog ade nylyl (beta,gamma-methylene)diphosphonate, 7,8-diaminononanoic acid, a nd Mn2+. One molecule of each substrate binds to one monomer of the en zyme, ADP and the ATP analogue bind to the classical mononucleotide bi nding fold with the phosphate groups close to the phosphate binding lo op Gly8-Thr16 between beta-strand beta 1 and the N-terminus of alpha-h elix alpha 1. The adenine ring is bound in a pocket between beta-stran ds beta 6 and beta 7. In the quaternary complex with Mn2+, the metal b inding site is found in the vicinity of the beta- and gamma-phosphate groups. Two oxygen atoms from the phosphates and oxygen atoms from the side chains of Asp54, Thr16, and Glu115 are ligands to the Mn2+ ion i n the quaternary complex. In the complex with ADP and the N7-carbamate of 7,8-diaminononanoic acid prepared in the presence of Ca2+ ions, a different metal binding site is found. The Ca2+ ion is coordinated to an oxygen atom of the alpha-phosphate group of the nucleotide, the sid e chain of Asp54, and solvent molecules. The 7,8-diaminononanoic acid substrate molecule interacts with residues from both subunits, making the dimer the minimal functional unit. The diamino group binds between the loops after beta 2 and beta 4, and the terminal carboxyl group at the hydrophobic tail of the substrate interacts with the amino termin us of helix alpha 5 and with the side chain of Tyr187 in helix alpha 6 of the second subunit at the monomer-monomer interface. Strong additi onal electron density close to the N7 nitrogen atom of the 7,8-diamino nonanoic acid substrate in some complexes indicates that, even in the absence of added bicarbonate in the crystallization mixture, the carba mylated intermediate is formed in the crystal. The crystal structure o f the binary complex of DTBS with the analog 3-(1-aminoethyl)nonanedio ic acid is identical to that of the binary complex with substrate, pro viding further evidence that the true reaction intermediate has formed in these crystals. The overall structures of the native enzyme and th e enzyme in the six complexes are very similar. However, binding of th e substrate and the mononucleotide causes local conformational changes of loop regions close to the active site. On the basis of the crystal structures of these complexes, a reaction mechanism for dethiobiotin synthetase is proposed.