Abstract
The LarA superfamily consists of nickel-dependent enzymes catalyzing racemization/epimerization reactions using a variety of α-hydroxy acids. The first-characterized LarA, a lactate racemase from Lactobacillus plantarum, led to the discovery of the nickel-pincer nucleotide (NPN) cofactor that is utilized by family members with alternative substrates, including malate racemase from Thermoanaerobacterium thermosaccharolyticum (Mar2). In this work, a higher resolution crystal structure of Mar2 was obtained with better data quality that revealed new structural and dynamic characteristics of the protein. A model of the Mar2 structure with bound cofactor and substrate was generated to uncover the common and the unique features among two distinct subgroups in the LarA superfamily. In addition, structure-guided mutational studies were used to examine the importance of residues that are modeled to interact with NPN and to explore which residues were critical for conferring specificity for malate. In particular, substitution of two residues involved in substrate binding in Mar2 to match the corresponding residues in LarA led to the acquisition of low levels of lactate racemase activity. Of additional interest, the substrate spectrum was expanded to include tartrate, an analog of malate. These new findings will help to better understand structure–function relationships of many other LarA homologs that are broadly distributed in bacterial and archaeal species.
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Acknowledgements
This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Use of the LS-CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). This work is supported by National Science Foundation Grant CHE 1807073 (to R.P.H. and J.H.), National Institutes of Health Grant GM128959 (to R.P.H. and J.H.), and Fonds de la Recherche Scientifique (FNRS) (to B.D. and J.U.). We thank Alessia Citti for her participation in the construction of the Mar2 variants.
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Gatreddi, S., Urdiain-Arraiza, J., Desguin, B. et al. Structural and mutational characterization of a malate racemase from the LarA superfamily. Biometals 36, 303–313 (2023). https://doi.org/10.1007/s10534-022-00372-x
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DOI: https://doi.org/10.1007/s10534-022-00372-x