Abstract
Dihydrodipicolinate reductase (DHDPR) is a key enzyme in the diaminopimelate- and lysine-synthesis pathways that reduces DHDP to tetrahydrodipicolinate. Although DHDPR uses both NADPH and NADH as a cofactor, the structural basis for cofactor specificity and preference remains unclear. Here, we report that Paenisporosarcina sp. TG-14 PaDHDPR has a strong preference for NADPH over NADH, as determined by isothermal titration calorimetry and enzymatic activity assays. We determined the crystal structures of PaDHDPR alone, with its competitive inhibitor (dipicolinate), and the ternary complex of the enzyme with dipicolinate and NADPH, with results showing that only the ternary complex had a fully closed conformation and suggesting that binding of both substrate and nucleotide cofactor is required for enzymatic activity. Moreover, NADPH binding induced local conformational changes in the N-terminal long loop (residues 34–59) of PaDHDPR, as the His35 and Lys36 residues in this loop interacted with the 2′-phosphate group of NADPH, possibly accounting for the strong preference of PaDHDPR for NADPH. Mutation of these residues revealed reduced NADPH binding and enzymatic activity, confirming their importance in NADPH binding. These findings provide insight into the mechanism of action and cofactor selectivity of this important bacterial enzyme.
Highlights
Sporulation is a major survival strategy for certain Gram-positive bacteria, including Bacillus and Clostridium species[1,2,3]
NADH did not bind to PaDHDPR; PaDHDPR had strong affinity for NADPH, with a Kd value of 37 μM (Fig. 2A), which was slightly lower than those reported for EcDHDPR (2.1 μM) and SaDHDPR (0.9 μM)[23,26,28]
Comparative structural analyses suggested that transition between the open and closed conformations in DHDPRs is determined by the binding of both substrate and nucleotide, given that only the DPA/NADPH-bound form of PaDHDPR showed a closed conformation
Summary
Sporulation is a major survival strategy for certain Gram-positive bacteria, including Bacillus and Clostridium species[1,2,3]. DAP is a component of peptidoglycan in the cell wall of many bacteria that forms cross-links with and thereby confers rigidity to the bacterial cell wall[14,15,16] These two pathways mutually regulate cellular activity through a feedback mechanism, with DPA inhibiting lysine synthesis[17,18]. The mechanisms associated with DHDPR discrimination between NADH and NADPH and the region and residues responsible for cofactor selectivity remain unknown. TG-14 (PaDHDPR) isolated from sediment-laden, stratified basal ice from Taylor glacier, McMurdo dry valley, Antarctica[29] This Gram-positive species is reportedly capable of surviving millions of years in the spore state[30,31,32]. We determined the crystal structures of unliganded PaDHDPR and the enzyme in complex with DPA alone or with NADPH and performed mutagenesis studies in order to gain insight into the structural basis for this cofactor preference
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
