Crystal structures of the archaeal UDP‐GlcNAc 2‐epimerase from <i>Methanocaldococcus jannaschii</i> reveal a conformational change induced by UDP‐GlcNAc

Chen, Sheng-Chia; Huang, Chi‐Hung; Yang, Changju; Liu, Jai-Shin; Kuan, Shu-Min; Chen, Yeh

doi:10.1002/prot.24516

Cited by 12 publications

(15 citation statements)

References 31 publications

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“…deviation in positions for all Cα is about 1.0Å. We refer in the following the structure of the ternary complex between MnaA, UDP and UDP-GlcNAc as the “closed” form [ 60 ], and other states of the protein, either in apo form or a binary complex with UDP, as the “opened” form, consistent with past structural characterization of the enzymes [ 60 ]. This difference between monomers is comparable to the differences with the structure of the E .…”

Section: Resultssupporting

confidence: 55%

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

et al. 2016

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Here we describe a chemical biology strategy performed in Staphylococcus aureus and Staphylococcus epidermidis to identify MnaA, a 2-epimerase that we demonstrate interconverts UDP-GlcNAc and UDP-ManNAc to modulate substrate levels of TarO and TarA wall teichoic acid (WTA) biosynthesis enzymes. Genetic inactivation of mnaA results in complete loss of WTA and dramatic in vitro β-lactam hypersensitivity in methicillin-resistant S. aureus (MRSA) and S. epidermidis (MRSE). Likewise, the β-lactam antibiotic imipenem exhibits restored bactericidal activity against mnaA mutants in vitro and concomitant efficacy against 2-epimerase defective strains in a mouse thigh model of MRSA and MRSE infection. Interestingly, whereas MnaA serves as the sole 2-epimerase required for WTA biosynthesis in S. epidermidis, MnaA and Cap5P provide compensatory WTA functional roles in S. aureus. We also demonstrate that MnaA and other enzymes of WTA biosynthesis are required for biofilm formation in MRSA and MRSE. We further determine the 1.9Å crystal structure of S. aureus MnaA and identify critical residues for enzymatic dimerization, stability, and substrate binding. Finally, the natural product antibiotic tunicamycin is shown to physically bind MnaA and Cap5P and inhibit 2-epimerase activity, demonstrating that it inhibits a previously unanticipated step in WTA biosynthesis. In summary, MnaA serves as a new Staphylococcal antibiotic target with cognate inhibitors predicted to possess dual therapeutic benefit: as combination agents to restore β-lactam efficacy against MRSA and MRSE and as non-bioactive prophylactic agents to prevent Staphylococcal biofilm formation.

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Section: Resultssupporting

confidence: 55%

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

et al. 2016

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“…61 Reports on the crystal structures of E. coli (a Gram-negative bacterium) RffE, 55 Bacillus anthracis (a Gram-positive bacterium) 62 and Methanocaldococcus jannaschii (archaea) 63 non-hydrolyzing UDP-GlcNAc 2-epimerases identified the substrate binding pocket and the critical amino acid residues that are involved in catalysis. The absence of NAD + in the crystal structures of these epimerases supported the observation that NAD + was not required for the activity of the enzyme.…”

Section: Resultsmentioning

confidence: 99%

“…62,63 In order to understand the substrate specificity of NmSacA, homology models were created from the three structures of UDP- and UDP-GlcNAc-bound proteins using SWISS-MODEL. 64 The B. anthracis model gave the best QMEAN4 score.…”

Section: Resultsmentioning

confidence: 99%

Characterizing non-hydrolyzing Neisseria meningitidis serogroup A UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase using UDP-N-acetylmannosamine (UDP-ManNAc) and derivatives

Zhang

Muthana

et al. 2016

Carbohydrate Research

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Neisseria meningitidis serogroup A non-hydrolyzing uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase (NmSacA) catalyzes the interconversion between UDP-GlcNAc and uridine 5′-diphosphate-N-acetylmannosamine (UDP-ManNAc). It is a key enzyme involved in the biosynthesis of the capsular polysaccharide [-6ManNAcα1-phosphate-]n of N. meningitidis serogroup A, one of the six serogroups (A, B, C, W-135, X, and Y) that account for most cases of N. meningitidis-caused bacterial septicemia and meningitis. N. meningitidis serogroup A is responsible for large epidemics in the developing world, especially in Africa. Here we report that UDP-ManNAc could be used as a substrate for C-terminal His6-tagged recombinant NmSacA (NmSacA-His6) in the absence of UDP-GlcNAc. NmSacA-His6 was activated by UDP-GlcNAc and inhibited by 2-acetamidoglucal and UDP. Substrate specificity study showed that NmSacA-His6 could tolerate several chemoenzymatically synthesized UDP-ManNAc derivatives as substrates although its activity was much lower than non-modified UDP-ManNAc. Homology modeling and molecular docking revealed likely structural determinants of NmSacA substrate specificity. This is the first detailed study of N. meningitidis serogroup A UDP-GlcNAc 2-epimerase.

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“…Each protomer contains two Rossmann-like domains. The binding of UDP-GlcNAc to an allosteric site stabilizes the closed, active conformation of the enzyme, whereas the open form is not active (17). Open and closed conformations are believed to affect the hydrolyzing enzyme activity as well, but so far the precise relationship remains unclear.…”

mentioning

confidence: 99%

The tetrameric structure of sialic acid–synthesizing UDP-GlcNAc 2-epimerase from Acinetobacter baumannii: A comparative study with human GNE

Lai

Hsieh

et al. 2018

Journal of Biological Chemistry

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Sialic acid presentation on the cell surface by some pathogenic strains of bacteria allows their escape from the host immune system. It is one of the major virulence factors. Bacterial biosynthesis of sialic acids starts with the conversion of UDP-GlcNAc to UDP and ManNAc by a hydrolyzing 2-epimerase. Here, we present the crystal structure of this enzyme, named NeuC, from The protein folds into two Rossmann-like domains and forms dimers and tetramers as does the epimerase part of the bifunctional UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE). In contrast to human GNE, which showed only the closed conformation, the NeuC crystals contained both open and closed protomers in each dimer. Substrate soaking changed the space group from C222 to P222 In addition to UDP, an intermediate-like ligand was seen bound to the closed protomer. The UDP-binding mode in NeuC was similar to that in GNE, although a few side chains were rotated away. NeuC lacks the CMP-Neu5Ac-binding site for allosteric inhibition of GNE. However, the two enzymes as well as other NeuC homologues (but not SiaA from ) appear to be common in tetrameric organization. The revised two-base catalytic mechanism may involve His-125 (Glu-134 in GNE), as suggested by mutant activity analysis.

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Crystal structures of the archaeal UDP‐GlcNAc 2‐epimerase from Methanocaldococcus jannaschii reveal a conformational change induced by UDP‐GlcNAc

Cited by 12 publications

References 31 publications

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

Chemical Genetic Analysis and Functional Characterization of Staphylococcal Wall Teichoic Acid 2-Epimerases Reveals Unconventional Antibiotic Drug Targets

Characterizing non-hydrolyzing Neisseria meningitidis serogroup A UDP-N-acetylglucosamine (UDP-GlcNAc) 2-epimerase using UDP-N-acetylmannosamine (UDP-ManNAc) and derivatives

The tetrameric structure of sialic acid–synthesizing UDP-GlcNAc 2-epimerase from Acinetobacter baumannii: A comparative study with human GNE

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