Structure and Mechanism of d-Glucosaminate-6-phosphate Ammonia-lyase: A Novel Octameric Assembly for a Pyridoxal 5′-Phosphate-Dependent Enzyme, and Unprecedented Stereochemical Inversion in the Elimination Reaction of a d-Amino Acid

Abstract

d-Glucosaminate-6-phosphate ammonia-lyase (DGL) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that produces 2-keto-3-deoxygluconate 6-phosphate (KDG-6-P) in the metabolism of d-glucosaminic acid by Salmonella enterica serovar typhimurium. We have determined the crystal structure of DGL by SAD phasing with selenomethionine to a resolution of 2.58 Å. The sequence has very low identity with most other members of the aminotransferase (AT) superfamily. The structure forms an octameric assembly as a tetramer of dimers that has not been observed previously in the AT superfamily. PLP is covalently bound as a Schiff base to Lys-213 in the catalytic dimer at the interface of two monomers. The structure lacks the conserved arginine that binds the α-carboxylate of the substrate in most members of the AT superfamily. However, there is a cluster of arginines in the small domain that likely serves as a binding site for the phosphate of the substrate. The deamination reaction performed in D2O gives a KDG-6-P product stereospecifically deuterated at C3; thus, the mechanism must involve an enamine intermediate that is protonated by the enzyme before product release. Nuclear magnetic resonance (NMR) analysis demonstrates that the deuterium is located in the pro-R position in the product, showing that the elimination of water takes place with inversion of configuration at C3, which is unprecedented for a PLP-dependent dehydratase/deaminase. On the basis of the crystal structure and the NMR data, a reaction mechanism for DGL is proposed.

His‐163 is a stereospecific proton donor in the mechanism of d‐glucosaminate‐6‐phosphate ammonia‐lyase

“…This likely occurs because the protonation of the enamine intermediate, released from the mutant enzyme, is now nonenzymatic and occurs in the solution after product release from the enzyme. These results provide support for our previous proposal, based on the X-ray crystal structure, that His-163 is a proton donor in the reaction mechanism [3].…”

“…More recently, we have determined the X-ray crystal structure of DGL, and we confirmed that His-162 and His-163 are located near the active site (Fig. 1) [3]. These histidine residues are strictly conserved in the sequences of DGL, even with relatively low sequence identity (Fig.…”

“…H163A DGL was prepared by PCR mutagenesis, expressed, and purified by Ni‐MAC, as previously described for wild‐type DGL [ 2 , 3 ]. Although we had speculated that His‐162 and His‐163 might be responsible for the binding of the unmodified enzyme to the Ni‐MAC column, we found that the H163A mutant enzyme behaved similarly on the column as wild‐type DGL.…”

“…Furthermore, the substrate concentration is 10 mM, while the enzyme concentration is 25 μM, so if it were enzyme bound, the intermediate would have a molar extinction coefficient of ≥56 000 M −1 Ácm −1 . No PLP enzyme intermediate is known, which has an absorbance at 293 nm with such From reference [3].…”

“…S1). We found previously that the reaction of DGL in D 2 O gives a single diastereomeric 3‐deutero‐KDG‐6‐P, where the pro‐ R proton of KDG‐6‐P is stereospecifically replaced by deuterium, resulting in an unusual inversion of stereochemistry in a β‐elimination reaction [ 3 ]. Thus, the unsaturated enamine intermediate must be protonated on the opposite face than water is eliminated from, possibly by conserved His‐163 as a proton donor.…”