Joseph Dmitri D A, Jiao Wanting, Kessans Sarah A, Parker Emily J
Biomolecular Interaction Centre and Department of Chemistry, University of Canterbury, Christchurch, New Zealand.
Proteins. 2014 Sep;82(9):2054-66. doi: 10.1002/prot.24558. Epub 2014 Apr 16.
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.
N-乙酰神经氨酸（NANA）是最常见的天然存在的唾液酸，在一些神经侵袭性细菌（如脑膜炎奈瑟菌）的发病机制中起关键作用。NANA在原核生物中通过磷酸烯醇丙酮酸和N-乙酰甘露糖胺之间的缩合反应合成。该反应由一种结构域交换的同二聚体酶N-乙酰神经氨酸合酶（NANAS）催化。NANAS由两个不同的结构域组成；一个N端催化（β/α）8桶状结构域与一个C端抗冻蛋白样（AFPL）结构域相连。我们通过对NmeNANAS的截短变体进行表征来研究AFPL结构域的作用，发现该变体可溶但无活性。使用分析超速离心和分析尺寸排阻法来探究NmeNANAS截短体的四级结构状态，结果表明AFPL结构域的缺失会使该酶的二聚体形式不稳定。这项研究的结果因此证明，AFPL结构域对NANAS的催化功能和四级结构稳定性都起着关键作用。小角X射线散射、分子动力学模拟和氨基酸取代揭示了一个复杂的氢键中继系统，该系统跨越亚基边界连接催化结构域和AFPL结构域的作用。
