Active Site Histidines Link Conformational Dynamics with Catalysis on Anti-Infective Target 1-Deoxy-d-xylulose 5-Phosphate Synthase

Abstract

The product of 1-deoxy-D-xyluose 5-phosphate (DXP) synthase, DXP, feeds into the bacterial biosynthesis of isoprenoids, thiamin diphosphate (ThDP), and pyridoxal phosphate. DXP is essential for human pathogens but not utilized by humans; thus, DXP synthase is an attractive antiinfective target. The unique ThDP-dependent mechanism and structure of DXP synthase offer ideal opportunities for selective targeting. Upon reaction with pyruvate, DXP synthase uniquely stabilizes the predecarboxylation intermediate, C2α-lactylThDP (LThDP), in a closed conformation. Subsequent binding of D-glyceraldehyde 3-phosphate induces an open conformation that is proposed to destabilize LThDP, triggering decarboxylation. Evidence for the closed and open conformations has been revealed by hydrogen-deuterium exchange mass spectrometry and X-ray crystallography, which indicate that H49 and H299 are involved in conformational dynamics and movement of the fork and spoon motifs away from the active site is important for the closedto-open transition. Interestingly, H49 and H299 are critical for DXP formation and interact with the predecarboxylation intermediate in the closed conformation. H299 is removed from the active *

First crystal structures of 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) from Mycobacterium tuberculosis indicate a distinct mechanism of intermediate stabilization

“…In both structures of Mt DXPS, Histidine 296, which corresponds to His304 in drDXPS, could not be observed. This His304 is part of the active site and has previously been identified by mutational studies as important for the catalysis through a stabilization of both LThDP and the closed conformation of the enzyme 19 , 26 . Experiments have demonstrated a 90% reduction of DXPS activity when His304 is mutated to alanine 32 .…”

“…DXPS catalyzes the ThDP-dependent decarboxylation of pyruvate and subsequent carboligation with d -glyceraldehyde-3-phosphate (D-GAP), the second substrate, following a preferred-order, random-sequential reaction mechanism (Fig. 1 ) 19 , 20 . Targeting this enzyme has the additional benefit of not only inhibiting the biosynthesis of the terpene precursors, but also the biosynthesis of the vitamins B1 and B6, which are synthesized from the product of DXPS, the branch-point metabolite 1-deoxy- d -xylulose 5-phosphate (DOXP) 21 .…”

“…In contrast, the DXPS enzyme is less explored—with just five protein structures published to date, four from D.radiodurans and one from E. coli 25 – 27 . The low number of crystal structures may be a consequence of the enzyme´s susceptibility to proteolytic degradation 25 , 27 , 28 or conformational changes during the catalytic cycle 19 , 29 —both properties that can make it difficult to obtain a homogeneous protein sample for crystallization. We recently published a modified drDXPS protein with improved crystallographic properties and speculated that our approach should be applicable to all DXPS homologues 27 .…”

Structure–function analyses of the G729R 2-oxoadipate dehydrogenase genetic variant associated with a disorder of l-lysine metabolism

“…A closer examination of the high-resolution MS signals revealed several peptides with bimodal mass envelope distributions that could be curveresolved into two closely spaced peaks, an indication of 'EX1 kinetics' (22). Unlike those peptides behaving according to 'EX2 kinetics', characterized by mass peaks gradually shifting to higher mass in MS-based analysis with peaks moving to the right, the spectrum of 'EX1 peptides' displayed two populations: a higher m/z envelope which has undergone an unfolding event (to the right) and a lower m/z envelope which hasn't (to the left in the deconvoluted spectrum) (23,24). Among the 'EX1 peptides' identified in the current study are peptides encompassing residues 256-271, 680-692 and 797-819 (see time course of HDX for the three peptides with side-by-side presentation of deconvoluted spectra for E1a and G729R E1a in Fig.…”

“…In summary, the following cross-linked Lys residues were identified on both E1a and G729R E1a: Lys 148 and Lys 188 from the N-terminal region; Lys 300 and Lys 450 from the ThDP and Mg 2+ binding region and Lys 818 , Lys 826/827 , Lys 852/854 from the C-terminal region. On E2o: the cross-linked Lys 24 (Tables S4, S5). Intriguingly, a great number of cross-links formed between the Lys residues from the C-terminal region of E1a and of G729R E1a, and those from the lipoyl domain of E2o have been detected, suggesting that the Cterminal region of E1a/G729R E1a could be important for interaction with E2o and for substrate channeling.…”