title: Cloning, expression, purification, crystallization and preliminary X-ray diffraction studies of N-acetylneuraminate lyase from methicillin-resistant Staphylococcus aureus.
abstract: The enzyme N-acetylneuraminate lyase (EC 4.1.3.3) is involved in the metabolism of sialic acids. Specifically, the enzyme catalyzes the retro-aldol cleavage of N-acetylneuraminic acid to form N-acetyl-D-mannosamine and pyruvate. Sialic acids comprise a large family of nine-carbon amino sugars, all of which are derived from the parent compound N-acetylneuraminic acid. In recent years, N-acetylneuraminate lyase has received considerable attention from both mechanistic and structural viewpoints and has been recognized as a potential antimicrobial drug target. The N-acetylneuraminate lyase gene was cloned from methicillin-resistant Staphylococcus aureus genomic DNA, and recombinant protein was expressed and purified from Escherichia coli BL21 (DE3). The enzyme crystallized in a number of crystal forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.70 Å resolution in space group P2₁. Molecular replacement indicates the presence of eight monomers per asymmetric unit. Understanding the structural biology of N-acetylneuraminate lyase in pathogenic bacteria, such as methicillin-resistant S. aureus, will provide insights for the development of future antimicrobials.
authors.affiliation: Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8041, New Zealand.
authors.forename: Rachel A
authors.initials: RA
authors.lastname: North
authors.forename: Sarah A
authors.initials: SA
authors.lastname: Kessans
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Atkinson
authors.forename: Hironori
authors.initials: H
authors.lastname: Suzuki
authors.forename: Andrew J A
authors.initials: AJ
authors.lastname: Watson
authors.forename: Benjamin R
authors.initials: BR
authors.lastname: Burgess
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Angley
authors.forename: André O
authors.initials: AO
authors.lastname: Hudson
authors.forename: Arvind
authors.initials: A
authors.lastname: Varsani
authors.forename: Michael D W
authors.initials: MD
authors.lastname: Griffin
authors.forename: Antony J
authors.initials: AJ
authors.lastname: Fairbanks
authors.forename: Renwick C J
authors.initials: RC
authors.lastname: Dobson
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D055624:Methicillin-Resistant Staphylococcus aureus / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D019158:N-Acetylneuraminic Acid / Q000737:chemistry / Q000378:metabolism; D007652:Oxo-Acid-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007652:Oxo-Acid-Lyases; C018728:N-acetylneuraminate lyase; D019158:N-Acetylneuraminic Acid
keywords: MRSA; N-acetylneuraminate lyase; NAL; Staphylococcus aureus; antibiotic resistance; sialic acid metabolism