Arg314 Is Essential for Catalysis by <i>N</i>-Acetyl Neuraminic Acid Synthase from <i>Neisseria meningitidis</i>

Joseph, Dmitri D. A.; Jiao, Wanting; Parker, Emily J.

doi:10.1021/bi400062c

Cited by 11 publications

(31 citation statements)

References 28 publications

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“…The striking result of these studies was the change in the affinity of the truncated protein for the reduced form of ManNAc (rManNAc). This substrate analogue binds to the wild‐type enzyme only in the presence of PEP . This observation is consistent with the known order of binding where PEP binds first to form the ManNAc binding site .…”

Section: Resultssupporting

confidence: 85%

“…The AFPL domain of Nme NANAS is a unique TIM barrel extension . It provides an essential arginine residue into the active site of the opposing monomer, which is involved in steering ManNAc into a reactive position with PEP . As observed in the crystal structure of Nme NANAS (Fig.…”

Section: Discussionmentioning

confidence: 89%

“…ITC data from G272Term were compared with previously determined dissociation constants ( K d ) for Nme NANAS wild‐type (Fig. ; Table ) …”

Section: Resultsmentioning

confidence: 99%

“…The affinity of substrate and metal ions for G272Term was assessed by ITC as previously described for wild‐type Nme NANAS . ITC experiments were run using a VP‐ITC unit operating at 298 K (MicroCal; GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

“…The AFPL domain caps the (βα) 8 barrel domain of the opposing monomer and contributes an essential arginine into the catalytic site . Arg314 of Nme NANAS has been determined to play an important role in steering ManNAc into an appropriate orientation for catalyzing reaction with PEP . While Arg314 plays an important part in the catalytic function of NANAS, the full purpose of the AFPL domain has not been determined.…”

Section: Introductionmentioning

confidence: 99%

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Substrate-mediated control of the conformation of an ancillary domain delivers a competent catalytic site forN-acetylneuraminic acid synthase

et al. 2014

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N-Acetylneuraminic acid (NANA) is the most common naturally occurring sialic acid and plays a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. NANA is synthesized in prokaryotes via a condensation reaction between phosphoenolpyruvate and N-acetylmannosamine. This reaction is catalyzed by a domain swapped, homodimeric enzyme, N-acetylneuraminic acid synthase (NANAS). NANAS comprises two distinct domains; an N-terminal catalytic (β/α)8 barrel linked to a C-terminal antifreeze protein-like (AFPL) domain. We have investigated the role of the AFPL domain by characterizing a truncated variant of NmeNANAS, which was discovered to be soluble yet inactive. Analytical ultracentrifugation and analytical size exclusion were used to probe the quaternary state of the NmeNANAS truncation, and revealed that loss of the AFPL domain destabilizes the dimeric form of the enzyme. The results from this study thereby demonstrate that the AFPL domain plays a critical role for both the catalytic function and quaternary structure stability of NANAS. Small angle X-ray scattering, molecular dynamics simulations, and amino acid substitutions expose a complex hydrogen-bonding relay, which links the roles of the catalytic and AFPL domains across subunit boundaries.

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Section: Resultssupporting

confidence: 85%

Section: Discussionmentioning

confidence: 89%

“…ITC data from G272Term were compared with previously determined dissociation constants ( K d ) for Nme NANAS wild‐type (Fig. ; Table ) …”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Substrate-mediated control of the conformation of an ancillary domain delivers a competent catalytic site forN-acetylneuraminic acid synthase

et al. 2014

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Flagellin glycosylation with pseudaminic acid in Campylobacter and Helicobacter: prospects for development of novel therapeutics

Ud-Din

Roujeinikova

2017

Cell. Mol. Life Sci.

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Many pathogenic bacteria require flagella-mediated motility to colonise and persist in their hosts. Helicobacter pylori and Campylobacter jejuni are flagellated epsilonproteobacteria associated with several human pathologies, including gastritis, acute diarrhea, gastric carcinoma and neurological disorders. In both species, glycosylation of flagellin with an unusual sugar pseudaminic acid (Pse) plays a crucial role in the biosynthesis of functional flagella, and thereby in bacterial motility and pathogenesis. Pse is found only in pathogenic bacteria. Its biosynthesis via six consecutive enzymatic steps has been extensively studied in H. pylori and C. jejuni. This review highlights the importance of flagella glycosylation and details structural insights into the enzymes in the Pse pathway obtained via a combination of biochemical, crystallographic, and mutagenesis studies of the enzyme-substrate and -inhibitor complexes. It is anticipated that understanding the underlying structural and molecular basis of the catalytic mechanisms of the Pse-synthesising enzymes will pave the way for the development of novel antimicrobials.

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Kinetic characterization of two neuraminic acid synthases and evaluation of their application potential

Çakar,

Milčić,

Andreadaki

et al. 2024

Appl Microbiol Biotechnol

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Neuraminic acid synthases are an important yet underexplored group of enzymes. Thus, in this research, we performed a detailed kinetic and stability analysis and a comparison of previously known neuraminic acid synthase from Neisseria meningitidis, and a novel enzyme, PNH 5 , obtained from a metagenomic library. A systematic analysis revealed a high level of similarity of PNH 5 to other known neuraminic acid synthases, except for its pH optimum, which was found to be at 5.5 for the novel enzyme. This is the first reported enzyme from this family that prefers an acidic pH value. The effect of different metal cofactors on enzyme activity, i.e. Co 2+ , Mn 2+ and Mg 2+ , was studied systematically. The kinetics of neuraminic acid synthesis was completely elucidated, and an appropriate kinetic model was proposed. Enzyme stability study revealed that the purified enzyme exhibits changes in its structure during time as observed by differential light scattering, which cause a drop in its activity and protein concentration. The operational enzyme stability for the neuraminic acid synthase from N. meningitidis is excellent, where no activity drop was observed during the batch reactor experiments. In the case of PNH 5 , some activity drop was observed at higher concentration of substrates. The obtained results present a solid platform for the future application of these enzymes in the synthesis of sialic acids. Key points• A novel neuraminic acid synthase was characterized.• The effect of cofactors on NeuS activity was elucidated.• Kinetic and stability characterization of two neuraminic acid synthases was performed.

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Arg314 Is Essential for Catalysis by N-Acetyl Neuraminic Acid Synthase from Neisseria meningitidis

Cited by 11 publications

References 28 publications

Substrate-mediated control of the conformation of an ancillary domain delivers a competent catalytic site forN-acetylneuraminic acid synthase

Substrate-mediated control of the conformation of an ancillary domain delivers a competent catalytic site forN-acetylneuraminic acid synthase

Flagellin glycosylation with pseudaminic acid in Campylobacter and Helicobacter: prospects for development of novel therapeutics

Kinetic characterization of two neuraminic acid synthases and evaluation of their application potential

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