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Structural, Functional and Calorimetric Investigation of MosA, a Dihydrodipicolinate Synthase from Sinorhizobium meliloti L5–30, does not Support Involvement in Rhizopine Biosynthesis
Author(s) -
Phenix Christopher P.,
Nienaber Kurt,
Tam Pui Hang,
Delbaere Louis T. J.,
Palmer David R. J.
Publication year - 2008
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200700569
Subject(s) - sinorhizobium meliloti , stereochemistry , schiff base , chemistry , active site , enzyme , biochemistry , mutant , gene
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 Å 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.