Direct Identification of Enzyme Active Site Residues by Solid-State REDOR NMR: Application to KDO8P Synthase

Abstract

In this communication we report the first direct identification of active site residues of the enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase (KDO8PS). This is accomplished by the application of rotational-echo double-resonance1 (REDOR) solid-state NMR experiments to lyophilized binary complexes of uniformly 15N-labeled KDO8PS with each of its natural substrates, phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate (A5P). Its complex with the mechanism-based inhibitor, 2 (Scheme 1), the most potent inhibitor known to date2 (Ki ) 0.4 μM), is studied as well and its binding is compared to that of the natural substrates. The REDOR technique1 is based on selective reintroduction of (magnetic) dipolar interactions between pairs of specific types of nuclei, and in favorable cases enables their accurate measurement from which distance constraints can be obtained.3 Its application for the study of protein-ligand interactions is achieved by incorporation of stable isotopes at known positions in the protein that are proximate to (labeled) ligand moieties. In previous studies, REDOR measured distances were shown to be in excellent agreement with X-ray data.3a,b Moreover, 31P{13C} REDOR was employed to investigate the conformation of the bound inhibitor glyphosate in the enzyme-substrate-inhibitor ternary complex of 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS),3c for which no X-ray structure is available. REDOR, e.g., 31P{13C} and 15N {31P}, was also used for the determination of structural constraints in the above ternary complex,3d and to directly monitor transient enzyme-intermediate complexes of EPSPS3e and of UDP-GlcNac enolpyruvoyl transferase (Mur Z).3f The enzyme KDO8PS catalyzes the condensation reaction between A5P and PEP to form KDO8P and inorganic phosphate (Pi, Scheme 1).4 This important enzymatic reaction plays a crucial role in the assembly process of lipopolysaccharides of most Gramnegative bacteria,5 and is therefore an attractive target for the design of novel antibacterial drugs. Interestingly, unlike most PEPutilizing enzymes that cleave the high-energy P-O bond of PEP, KDO8PS and its family members (Mur Z, EPSPS, and 3-deoxyD-arabino-heptulosonate-7-phosphate synthase6 (DAHPS)) cleave the C-O bond of PEP.7 While the mechanisms of EPSPS8a (targeted by the herbicide glyphosate) and Mur Z8b (targeted by the antibiotic fosfomycin) were unambiguously characterized, the mechanisms of KDO8PS and DAHPS continue to be uncertain. Recent studies9a,2 on KDO8PS, including the synthesis and evaluation of the first acyclic bisubstrate inhibitor (2),2 supported a mechanism involving the formation of an acyclic bisphosphate intermediate 1 (Scheme 1).9b Mutagenesis studies10 identified in E. coli KDO8PS two vital cysteines, Cys38 and Cys166, yet their specific role still remains unclear. In addition, since to date neither the crystal structure nor the identity of the active site residues of KDO8PS are known, rational design of potent inhibitors for this enzyme is impaired. As part of our study on the structurefunction relationship of KDO8PS, [u-15N]KDO8PS was prepared and interactions between 15N enzyme labels to 31P nuclei of the ligands were examined by REDOR NMR.11 The 15N NMR CPMAS echo spectrum of [u-15N]KDO8PS binary complex with PEP (Figure 1a) shows resolved peaks for the nitrogen atoms of 10 side-chain arginine (guanidino: internal , 60 ppm; terminal η, 49 ppm), 22 lysine ( 9.0 ppm) residues, and an intense amide peak (95 ppm). Peaks of six side-chain histidines (δ1 224; 2 143 ppm) are not shown. In addition, detailed inspection of this spectrum reveals fine structure for the arginine (56.0 ppm), and a high field shoulder for lysine (3.0 ppm), attributed to a single arginine and to a single lysine residue residing in a distinct chemical environment. Interestingly, these two peaks are also present in the 15N CPMAS reference spectra of the other two complexes, KDO8PS-A5P and KDO8PS-2 (spectra not shown), implying that they arise from chemically distinct environments inherent to the enzyme itself, and are therefore not a result of interaction with a specific ligand. As will be shown below, these residues will be associated with specific recognition and binding of PEP by the enzyme.