X-ray crystallography–based structural elucidation of enzyme-bound intermediates along the 1-deoxy-d-xylulose 5-phosphate synthase reaction coordinate

Chen, Percival Yang-Ting; DeColli, Alicia A.; Meyers, Caren L. Freel; Drennan, Catherine L.

doi:10.1074/jbc.ra119.009321

Cited by 35 publications

(105 citation statements)

References 48 publications

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“…We have recently reported the first ligand-bound crystal structures of Deinococcus radiodurans DXP synthase that provide further support for this hypothesis. 16 Consistent with mutagenesis results, 19,21,22 the structure of DXP synthase with PLThDP bound shows that H51 and H304 (analogous to E. coli DXP synthase residues H49 and H299, respectively) are within hydrogen bonding distance of the phosphonyl group of PLThDP in the closed conformation (Figure 2A,C). By extension, H51 and H304 are predicted to interact with and stabilize the carboxyl group of the predecarboxylation intermediate LThDP.…”

Section: Graphical Abstractsupporting

confidence: 80%

“…Previous kinetic analyses of H49 and H299 variants (and analogous variants on D. radiodurans DXP synthase) were conducted under aerobic conditions, 19,21 prior to the discovery that oxygenase activity interferes in mechanistic and structural studies of DXP synthase. 13,16 Here, we show that all H49 and H299 variants display significant, albeit inefficient, oxygenase activity ( Figure S1); accordingly, we conducted detailed kinetic analyses under anaerobic conditions, to exclude any potentially confounding effects of oxidative pyruvate decarboxylation in comparing kinetic parameters for H49A, H49N, H299A, H299N, and wild type DXP synthase. Variants were selected to determine the effects of both conservative (asparagine) and nonconservative (alanine) substitutions on substrate affinity, catalytic activity, and inhibition under anaerobic conditions.…”

Section: Steady State Characterization Of H49 and H299 Variantsmentioning

confidence: 97%

“…23 MAP forms the LThDP-like intermediate PLThDP, which cannot undergo decarboxylation, and is thus a useful probe for interrogating steps prior to LThDP decarboxylation. 24,25 To provide biochemical evidence of the roles of H49 and H299 in MAP binding, suggested by the PLThDP-bound structure of DXP synthase, 16 we determined the effects of substituting H49 and H299 on K i MAP ( Table 2 and Figure S4). (Table 2).…”

Section: Characterization Of Dxp Synthase Inhibition By Mapmentioning

confidence: 99%

“…The relative abundance of these envelopes depends upon the ligand state of DXP synthase. 18 In the presence of MAP, this 16 Importantly, the movement of the fork (including H299) and spoon motifs away from the active site results in the change in solvent accessibility of the EX1 regions.…”

Section: Conformational Equilibrium Of Histidine Variants Detected Bymentioning

confidence: 99%

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

et al. 2019

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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 *

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Section: Graphical Abstractsupporting

confidence: 80%

Section: Steady State Characterization Of H49 and H299 Variantsmentioning

confidence: 97%

Section: Characterization Of Dxp Synthase Inhibition By Mapmentioning

confidence: 99%

Section: Conformational Equilibrium Of Histidine Variants Detected Bymentioning

confidence: 99%

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

et al. 2019

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“…The aliphatic index was 88.74, and the grand average of hydropathicity (GRAVY) was −0.113. The X‐ray crystal structure of DXS enzyme‐bound intermediates has been elucidated before (Chen et al ), so we chose the 1‐deoxy‐ d ‐xylulose‐5‐phosphate synthase 6ouv.1 (sequence identity 40.42%, X‐ray, 1.9 Å) for homology modeling to construct a three‐dimensional structure of TwDXS (Fig. B).…”

Section: Resultsmentioning

confidence: 99%

The expression of TwDXS in the MEP pathway specifically affects the accumulation of triptolide

Zhang

Zhao

Wang

et al. 2019

Physiologia Plantarum

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1‐Deoxy‐d‐xylulose‐5‐phosphate synthase (DXS) is the first enzyme in the plant 2‐C‐methyl‐d‐erythritol 4‐phosphate (MEP) pathway of terpenoid synthesis. TwDXS is a prominent protein in the Tripterygium wilfordii proteome, with especially high expression in the root periderm. It is significantly regulated by methyl jasmonate. Here, we studied the influence of TwDXS expression on bioactive terpenoids in T. wilfordii. Specific fragments of TwDXS (GenBank: AKP20998.1) with lengths of 2148 and 437 bp were amplified to construct the overexpression (OE) and RNA‐interference (RNAi) vectors, respectively. After transformation of suspension cells, the expression of TwDXS and genes related to the terpenoid biosynthetic pathway was measured using qRT‐PCR. TwDXS mRNA level was 153 and 43% of the control in the OE and RNAi lines. Related genes in the 2‐C‐methyl‐d‐erythritol 4‐phosphate (MEP), mevalonic acid (MVA) and downstream pathways showed similar trends to the changes of TwDXS expression. Ultra Performance Liquid Chromatography (UPLC) was employed to measure the accumulation of terpenoids. Importantly, the triptolide content showed significant differences in both the TwDXS OE (222.35% of the control) and RNAi (34.86% of the control). However, there were no obvious changes in the celastrol content. In this study, we verified that the expression of TwDXS affects triptolide but not celastrol in T. wilfordii via both TwDXS OE and RNAi experiments.

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Identification and Characterization of Thiamine Diphosphate‐Dependent Lyases with an Unusual CDG Motif

Lanza,

Rabe von Pappenheim,

Bjarnesen

et al. 2024

Angewandte Chemie

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The thiamine diphosphate (ThDP)‐binding motif, characterized by the canonical GDG(X)24‐27N sequence, is highly conserved among ThDP‐dependent enzymes. We investigated a ThDP‐dependent lyase (JanthE from Janthinobacterium sp. HH01) with an unusual cysteine (C458) replacing the first glycine of this motif. We found that JanthE has a high substrate promiscuity accepting long aliphatic α‐keto acids as donors. Sterically hindered aromatic aldehydes or non‐activated ketones are acceptor substrates, giving access to a variety of secondary and tertiary alcohols as carboligation products. The crystal structure solved at a resolution of 1.9 Å reveals that C458 is not primarily involved in the cofactor binding as previously thought for the canonical glycine. Instead, it coordinates methionine 406, thus ensuring the integrity of the active site and the enzyme activity. We further determined the long‐sought genuine tetrahedral intermediates formed with pyruvate and 2‐oxo‐butyrate in the pre‐decarboxylation states and unravel atomic details for their stabilization in the active site. Collectively, we unravel an unexpected role for the first residue of the ThDP‐binding motif and unlock a family of lyases able to perform valuable carboligation reactions.

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X-ray crystallography–based structural elucidation of enzyme-bound intermediates along the 1-deoxy-d-xylulose 5-phosphate synthase reaction coordinate

Cited by 35 publications

References 48 publications

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

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

The expression of TwDXS in the MEP pathway specifically affects the accumulation of triptolide

Identification and Characterization of Thiamine Diphosphate‐Dependent Lyases with an Unusual CDG Motif

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