Structural, Functional and Calorimetric Investigation of MosA, a Dihydrodipicolinate Synthase from Sinorhizobium meliloti L5–30, does not Support Involvement in Rhizopine Biosynthesis

Abstract

MosA is an enzyme from Sinorhizobium meliloti L5-30, a beneficial soil bacterium that forms a symbiotic relationship with leguminous plants. MosA was proposed to catalyze the conversion of scyllo-inosamine to 3-O-methyl-scyllo-inosamine (compounds known as rhizopines), despite the MosA sequence showing a strong resemblance to dihydrodipicolinate synthase (DHDPS) sequences rather than to methyltransferases. Our laboratory has already shown that MosA is an efficient catalyst of the DHDPS reaction. Here we report the structure of MosA, solved to 1.95 A resolution, which resembles previously reported DHDPS structures. In this structure Lys161 forms a Schiff base adduct with pyruvate, consistent with the DHDPS mechanism. We have synthesized both known rhizopines and investigated their ability to interact with MosA in the presence and absence of methyl donors. No MosA-catalyzed methyltransferase activity is observed in the presence of scyllo-inosamine and S-adenosylmethionine (SAM). 2-Oxobutyrate can form a Schiff base with MosA, acting as a competitive inhibitor of MosA-catalyzed dihydrodipicolinate synthesis. It can be trapped on the enzyme by reaction with sodium borohydride, but does not act as a methyl donor. The presence of rhizopines does not affect the kinetics of dihydrodipicolinate synthesis. Isothermal titration calorimetry (ITC) shows no apparent interaction of MosA with rhizopines and SAM. Similar experiments with pyruvate as titrant demonstrate that the reversible Schiff base formation is largely entropically driven. This is the first use of ITC to study Schiff base formation between an enzyme and its substrate.

Biochemical studies and crystal structure determination of dihydrodipicolinate synthase from Pseudomonas aeruginosa

“…The orientations of Thr44 and Tyr107 are well conserved as in the structure of PsDHDPS observed in other DHDPS structures [9,13,16, PDB ID: 3PUL, 3GOS]. However, the plane of the side chain of Tyr133 is found to have been rotated by more than 46 • with respect to the plane of the corresponding residue in other DHDPS structures [9,13,16, PDB ID: 3PUL, 3GOS]. The orientation of Lys161 is identical in all these structures while the orientation of side chain of Arg138 shows considerable variations in the DHDPS structures from various species.…”

“…The active site consisting of residues, Thr44, Tyr107 and Tyr133 which are stereochemically well arranged with hydrogen bonds of 2.6Å and 3.2Å between Thr44 and Tyr107, and Thr44 and Tyr133, respectively. The orientations of Thr44 and Tyr107 are well conserved as in the structure of PsDHDPS observed in other DHDPS structures [9,13,16, PDB ID: 3PUL, 3GOS]. However, the plane of the side chain of Tyr133 is found to have been rotated by more than 46 • with respect to the plane of the corresponding residue in other DHDPS structures [9,13,16, PDB ID: 3PUL, 3GOS].…”

“…As a result, the structure of PsDHDPS is observed only as a dimer. It may also be noted that the dimers of dimeric PsDHDPS are held more firmly than the corresponding dimers of tetrameric DHDPS structures [9,13,16, PDB ID: 3PUL, 3GOS].…”

“…In the proximity of active site, Lys161 was found to form a Schiff base with the first substrate, pyruvate. This arrangement is conserved in DHDPS enzymes from various species [9,13,16 [9,13,16, PDB ID: 3PUL, 3GOS], Tyr107 is projected towards the active site of the dimeric partner for completing the specifications of the active site triad in both molecules. It is noteworthy that Arg138 is situated at the entrance of the active site which is considered to promote the substrate Fig.…”

“…aeruginosa (PsDHDPS) shows a considerable variation in the amino acid sequence identities of DHDPS from other bacterial species having maximum identity of 60% with Acinetobacter baumannii (AbDHDPS) [PDB ID: 3PUL]. Although the crystal structures of DHDPS from several other organisms such as Escherichia coli (EcDHDPS) [9], Mycobacterium tuberculosis (MtDHDPS) [10], Corynebacterium glutamicum (CgDHDPS) [11], Staphylococcus aureus (SaDHDPS) [12], Neisseria meningitidis (NmDHDPS) [13], Bacillus anthracis (BaDHDPS) [14], Streptococcus pneumonia (SpDHDPS) [15], Synorhizobium melioti (SmDHDPS) [16], Methanocaldococcus jannaschii (MjDHDPS) [17], Thermotoga meritima (TmDHDPS) [18], and Bacilus clausii (BcDHDPS) [PDB ID: 3E96] have been reported but due to considerable structural and functional variations [9,12] it is essential to determine the structure of DHDPS proteins from various species so that a comprehensive understanding of the stereochemical details of the target enzyme could be achieved so that new molecules could be designed to inhibit the function of DHDPS. We report here cloning, expression, biochemical and biophysical characterizations as well as the determination of threedimensional structures of native enzyme PsDHDPS and its complex with lysine molecule.…”