Hager Fiona F, López-Guzmán Arturo, Krauter Simon, Blaukopf Markus, Polter Mathias, Brockhausen Inka, Kosma Paul, Schäffer Christina
NanoGlycobiology Unit, Department of NanoBiotechnology, Universität für Bodenkultur Wien, Vienna, Austria.
Division of Organic Chemistry, Department of Chemistry, Universität für Bodenkultur Wien, Vienna, Austria.
Front Microbiol. 2018 Jun 27;9:1356. doi: 10.3389/fmicb.2018.01356. eCollection 2018.
Various mechanisms of protein cell surface display have evolved during bacterial evolution. Several Gram-positive bacteria employ S-layer homology (SLH) domain-mediated sorting of cell-surface proteins and concomitantly engage a pyruvylated secondary cell-wall polymer as a cell-wall ligand. Specifically, pyruvate ketal linked to β-D-ManNAc is regarded as an indispensable epitope in this cell-surface display mechanism. That secondary cell wall polymer (SCWP) pyruvylation and SLH domain-containing proteins are functionally coupled is supported by the presence of an ortholog of the predicted pyruvyltransferase CsaB in bacterial genomes, such as those of and . The SCWP, consisting of pyruvylated disaccharide repeats [→4)-β-D-GlcNAc-(1→3)-4,6-Pyr-β-D-ManNAc-(1→] serves as a model to investigate the widely unexplored pyruvylation reaction. Here, we reconstituted the underlying enzymatic pathway in combination with synthesized compounds, used mass spectrometry, and nuclear magnetic resonance spectroscopy for product characterization, and found that CsaB-catalyzed pyruvylation of β-D-ManNAc occurs at the stage of the lipid-linked repeat. We produced the TagA (PAV_RS07420) and CsaB (PAV_RS07425) enzymes as recombinant, tagged proteins, and using a synthetic 11-phenoxyundecyl-diphosphoryl-α-GlcNAc acceptor, we uncovered that TagA is an inverting UDP-α-D-ManNAc:GlcNAc-lipid carrier transferase, and that CsaB is a pyruvyltransferase, with synthetic UDP-α-D-ManNAc and phosphoenolpyruvate serving as donor substrates. Next, to substitute for the UDP-α-D-ManNAc substrate, the recombinant UDP-GlcNAc-2-epimerase MnaA (PAV_RS07610) of was included in this reconstitution system. When all three enzymes, their substrates and the lipid-linked GlcNAc primer were combined in a one-pot reaction, a lipid-linked SCWP repeat precursor analog was obtained. This work highlights the biochemical basis of SCWP biosynthesis and bacterial pyruvyl transfer.
在细菌进化过程中，蛋白质细胞表面展示的多种机制不断演变。几种革兰氏阳性细菌利用S层同源性（SLH）结构域介导细胞表面蛋白的分选，并同时利用一种丙酮酸化的次生细胞壁聚合物作为细胞壁配体。具体而言，与β-D-甘露糖胺连接的丙酮酸缩酮被视为这种细胞表面展示机制中不可或缺的表位。细菌基因组中预测的丙酮酸转移酶CsaB直系同源物的存在，如[具体细菌名称1]和[具体细菌名称2]的基因组，支持了次生细胞壁聚合物（SCWP）丙酮酸化与含SLH结构域的蛋白质在功能上的耦合。由丙酮酸化二糖重复序列[→4)-β-D-葡糖胺-(1→3)-4,6-吡喃-β-D-甘露糖胺-(1→]组成的SCWP可作为研究广泛未被探索的丙酮酸化反应的模型。在此，我们结合合成化合物重建了潜在的酶促途径，使用质谱和核磁共振光谱对产物进行表征，发现CsaB催化的β-D-甘露糖胺丙酮酸化发生在脂连接重复序列阶段。我们将TagA（PAV_RS07420）和CsaB（PAV_RS07425）酶作为重组的、带标签的蛋白质进行表达，并且使用合成的11-苯氧基十一烷基二磷酸化-α-葡糖胺受体，我们发现TagA是一种构型翻转的UDP-α-D-甘露糖胺：葡糖胺脂载体转移酶，而CsaB是一种丙酮酸转移酶，合成的UDP-α-D-甘露糖胺和磷酸烯醇丙酮酸作为供体底物。接下来，为了替代UDP-α-D-甘露糖胺底物，将[具体细菌名称3]的重组UDP-葡糖胺-2-表异构酶MnaA（PAV_RS07610）纳入该重建系统。当将所有三种酶、它们的底物和脂连接的葡糖胺引物在一锅反应中混合时，获得了一种脂连接的SCWP重复序列前体类似物。这项工作突出了SCWP生物合成和细菌丙酮酸转移的生化基础。
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