Kinetic and structural studies of the reaction of Escherichia coli dihydrodipicolinate synthase with (S)‐2‐bromopropionate

• Published in: Acta crystallographica. Section D, Biological crystallography. Biological crystallography., 2022-07, Vol.78 (7), p.846-852
• Main Authors: Chooback, Lilian, Thomas, Leonard N., Blythe, Nathan, Karsten, William
• Format: Article
• Language: English
• Subjects: (S)‐2‐bromopropionate; Bromine; Carbonyls; Covalence; Crystal structure; crystallography; Crystals; Dihydrodipicolinate synthase; E coli; enzyme inhibition; enzyme structure; Enzymes; Escherichia coli; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - metabolism; Hydro-Lyases - chemistry; Hydro-Lyases - metabolism; Imines - metabolism; Kinetics; Lysine; lysine biosynthesis; Nucleophiles; Polyethylene glycol; Propionates - metabolism; Propionic acid; Pyruvates - chemistry; Pyruvates - metabolism; Pyruvic acid; Research Papers; Spermidine
• ISSN: 2059-7983; 0907-4449; 2059-7983; 1399-0047
• DOI: 10.1107/S2059798322005125

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine‐biosynthetic pathway converting pyruvate and l‐aspartate‐β‐semialdehyde to dihydrodipicolinate. Kinetic studies indicate that the pyruvate analog (S)‐2‐bromopropionate inactivates the enzyme in a pseudo‐first‐order process. An initial velocity pattern indicates that (S)‐2‐bromopropionate is a competitive inhibitor versus pyruvate, with an inhibition constant of about 8 mM. Crystals of DHDPS complexed with (S)‐2‐bromopropionate formed in a solution consisting of 50 mM HEPES pH 7.5, 18% polyethylene glycol 3350, 8 mM spermidine, 0.2 M sodium tartrate and 5.0 mg ml−1 DHDPS. The crystals diffracted to 2.15 Å resolution and belonged to space group P1. The crystal structure confirms the displacement of bromine and the formation of a covalent attachment between propionate and Lys161 at the active site of the enzyme. Lys161 is the active‐site nucleophile that attacks the carbonyl C atom of pyruvate and subsequently generates an imine adduct in the first half‐reaction of the ping‐pong enzymatic reaction. A comparison of the crystal structures of DHDPS complexed with pyruvate or (S)‐2‐bromopropionate indicates the covalent adduct formed from (S)‐2‐bromopropionate leads to a rotation of about 180° of the β–δ C atoms of Lys61 that aligns the covalently bound propionate fairly closely with the imine adduct formed with pyruvate. The crystal structure of dihydrodipicolinate synthase modified with (S)‐2‐bromopropionate at Lys161 is reported.