Crystal Structure of Shikimate Kinase from Mycobacterium tuberculosis Reveals the Dynamic Role of the LID Domain in Catalysis

Gu, Yijun; Reshetnikova, L.; Li, Yue; Wu, Yan; Yan, Honggao; Singh, Shivendra Kumar; Ji, Xinhua

doi:10.1016/s0022-2836(02)00339-x

Cited by 67 publications

(140 citation statements)

References 33 publications

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“…The strand order of the β-sheets is β13, β8, β7, β6, β1, β5, β2, β4, β3, and β9, β8, β13, β12, β10, β11 for the N-and Cterminal domains, respectively, with β8 and β13 linking the two domains. These features distinguish LpxK from other P-loop kinases, which usually consist of single Rossmann-fold domain with an internal five-stranded β-sheet containing the strand order β2, β3, β1, β4, β5 (19)(20)(21)(22). The active site Walker A/P-loop and Walker B motifs are located on the N-terminal domain in a pocket formed between the two domains ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…2A). As found in the majority of P-loop kinases, the Walker A motif is located between the first β-strand (β1) and the following α-helix (α2) of the polypeptide chain (19)(20)(21)(22). Even though all residues of LpxK were successfully modeled, loops encompassing residues 74-79 (L1), 234-236 (L2), and 261-262 (L3) displayed weaker density, with an increased average B-factor (128.0 Å 2 compared with 43.8 Å 2 for the entire polypeptide), indicating a higher degree of flexibility in these regions ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Bringing ATP close to the anionic phospholipids of the inner membrane may require additional interactions provided by the LpxK C-terminal domain to lower the free energy of nucleotide binding enough to overcome charge repulsion at the membrane surface. In most P-loop kinases, additions to the core α/β/α domain tend to be more subtle, often consisting of only a few secondary-structure elements, and these proteins implement surface clefts and dimerization to bind ATP and exclude water from the active site (21,22,31). Our crystal structures, along with gel-filtration analysis of the purified enzyme (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…First, only LpxK incubated with ATP/Mg 2+ crystallized with ADP and Mg 2+ in the active site, indicating that ATPase activity may have been achieved in the absence of DSMP during crystallization. Second, in other P-loop kinases, the Walker A hydroxyl-containing residue (T52 in LpxK), which follows the P-loop lysine, is often found to be directly involved in Mg 2+ binding (19)(20)(21)(22) and is sometimes assisted by a Walker B carboxylate (19,20,31,40). The precatalytic conformation of ATP/Mg 2+ -bound LpxK may be well-represented by the GTPγS/Mg 2+ -bound model of LpxK's closest structural homolog HypB, in which the γ-phosphate occupies an analogous position to the Mg 2+ ion of the LpxK structure (Fig.…”

Section: Lpxk Mutantmentioning

confidence: 99%

“…S1) (17)(18)(19)(20). Although crystal structures of a handful of other P-loop kinases have been determined previously (19,21,22), LpxK is at least 100 aa longer than the majority of these family members and is the only known membrane-bound lipid kinase in the group, thus making its study important for expanding our knowledge of both lipid kinases and the diverse P-loop kinase family. Purification and characterization of E. coli LpxK has been hampered because of instability of the enzyme (11).…”

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confidence: 99%

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Crystal structure of LpxK, the 4′-kinase of lipid A biosynthesis and atypical P-loop kinase functioning at the membrane interface

Emptage¹,

Daughtry²,

Pemble

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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In Gram-negative bacteria, the hydrophobic anchor of the outer membrane lipopolysaccharide is lipid A, a saccharolipid that plays key roles in both viability and pathogenicity of these organisms. The tetraacyldisaccharide 4′-kinase (LpxK) of the diverse P-loopcontaining nucleoside triphosphate hydrolase superfamily catalyzes the sixth step in the biosynthetic pathway of lipid A, and is the only known P-loop kinase to act upon a lipid substrate at the membrane. Here, we report the crystal structures of apo-and ADP/ Mg 2+ -bound forms of Aquifex aeolicus LpxK to a resolution of 1.9 Å and 2.2 Å, respectively. LpxK consists of two α/β/α sandwich domains connected by a two-stranded β-sheet linker. The N-terminal domain, which has most structural homology to other family members, is responsible for catalysis at the P-loop and positioning of the disaccharide-1-phosphate substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate and Mg 2+ cation using a 25°hinge motion about its base. Activity was severely reduced in alanine point mutants of conserved residues D138 and D139, which are not directly involved in ADP or Mg 2+ binding in our structures, indicating possible roles in phosphoryl acceptor positioning or catalysis. Combined structural and kinetic studies have led to an increased understanding of the enzymatic mechanism of LpxK and provided the framework for structurebased antimicrobial design.lipid metabolism | endotoxin | integral monotopic protein | Walker motif

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Lpxk Mutantmentioning

confidence: 99%

mentioning

confidence: 99%

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Crystal structure of LpxK, the 4′-kinase of lipid A biosynthesis and atypical P-loop kinase functioning at the membrane interface

Emptage¹,

Daughtry²,

Pemble

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Recombinant AroL‐Catalyzed Phosphorylation for the Efficient Synthesis of Shikimic Acid 3‐Phosphate

Schoenenberger¹,

Wszołek²,

Meier³

et al. 2018

Biotechnology Journal

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Shikimic acid 3-phosphate, as a central metabolite of the shikimate pathway, is of high interest as enzyme substrate for 5-enolpyruvoyl-shikimate 3-phosphate synthase, a drug target in infectious diseases and a prime enzyme target for the herbicide glyphosate. As the important substrate shikimic acid 3-phosphate is only accessible via a chemical multi-step route, a new straightforward preparative one-step enzymatic phosphorylation of shikimate using a stable recombinant shikimate kinase has been developed for the selective phosphorylation of shikimate in the 3-position. Highly active shikimate kinase is produced by straightforward expression of a synthetic aroL gene in Escherichia coli. The time course of the shikimate kinase-catalyzed phosphorylation is investigated by H- and P-NMR, using the phosphoenolpyruvate/pyruvate kinase system for the regeneration of the ATP cofactor. This enables the development of a quantitative biocatalytic 3-phosphorylation of shikimic acid. After a standard workup procedure, a good yield of shikimic acid 3-phosphate, with high HPLC- and NMR purity, is obtained. This efficient biocatalytic synthesis of shikimic acid 3-phosphate is superior to any other method and has been successfully scaled up to multi-gram scale.

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Study of the Phosphoryl‐Transfer Mechanism of Shikimate Kinase by NMR Spectroscopy

Prado

Lence

Vallejo

et al. 2016

Chemistry A European J

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The phosphoryl-transfer mechanism of shikimate kinase from Mycobacterium tuberculosis and Helicobacter pylori, which is an attractive target for antibiotic drug discovery, has been studied by 1D (1)H and (31)P NMR spectroscopy. Metaphosphoric acid proved to be a good mimetic of the metaphosphate intermediate and facilitated the ready and rapid evaluation by NMR spectroscopic analysis of a dissociative mechanism. The required closed form of the active site for catalysis was achieved by the use of ADP (product) or two synthetic ADP analogues (AMPNP, AMPCP). Molecular dynamics simulation studies reported here also revealed that the essential arginine (Arg116/Arg117 in H. pylori and M. tuberculosis, respectively), which activates the γ-phosphate group of ATP for catalysis and triggers the release of the product for turnover, would also be involved in the stabilisation of the metaphosphate intermediate during catalysis. We believe that the studies reported here will be helpful for future structure-based design of inhibitors of this attractive target. The approach is also expected be useful for studies on the possible dissociative mechanism of other kinase enzymes.

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Crystal Structure of Shikimate Kinase from Mycobacterium tuberculosis Reveals the Dynamic Role of the LID Domain in Catalysis

Cited by 67 publications

References 33 publications

Crystal structure of LpxK, the 4′-kinase of lipid A biosynthesis and atypical P-loop kinase functioning at the membrane interface

Crystal structure of LpxK, the 4′-kinase of lipid A biosynthesis and atypical P-loop kinase functioning at the membrane interface

Recombinant AroL‐Catalyzed Phosphorylation for the Efficient Synthesis of Shikimic Acid 3‐Phosphate

Study of the Phosphoryl‐Transfer Mechanism of Shikimate Kinase by NMR Spectroscopy

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