PelX is a UDP-N-acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation
PelX is a UDP-N-acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation
复制标题
DOI：
10.1074/jbc.ra120.014555
复制
发表时间：
2020-08-21
影响因子：
4.8
通讯作者：
Howell, P. Lynne
中科院分区：
文献类型：
--
作者：
Marmont, Lindsey S.;Whitfield, Gregory B.;Howell, P. Lynne
Pel是一种富含GalNAc的细菌多糖，对铜绿假单胞菌生物膜的结构和功能有重要作用。Pel ABCDEFG操纵子在不同的细菌物种中高度保守，因此Pel可能是一种广泛存在的生物膜决定因子。先前对pelX基因簇的注释帮助我们鉴定了一个额外的基因pelX，它存在于> 100种不同细菌物种中的pelABCDEFG附近。ThepelX基因被预测为编码短链脱氢酶/还原酶（SDR）超家族的成员，但其在Pel依赖性生物膜形成中的潜在作用尚不清楚。在此，我们使用假单胞菌蛋白Pf-5作为模型来阐明PelX功能，因为假单胞菌在其spel基因簇中缺少apelX同源物。我们发现P。蛋白质形成Pel依赖性生物膜;然而，尽管在这些条件下表达pelX，生物膜形成在Delta pelX菌株中不受影响。这一观察结果使我们鉴定出apelXparaX，PFL_5533，我们在此将其命名为PgnE，其似乎是功能冗余的topelX。与此一致，Δ pelX Δ pgnEdouble突变体在其形成Pel依赖性生物膜的能力方面实质上受损。为了理解这一观察的分子基础，我们将PelX的结构确定为2.1埃分辨率。结构显示PelX类似于UDP-GlcNAc C4-差向异构酶。使用（1）H NMR分析，我们表明PelX催化UDP-GlcNAc和UDP-GalNAc之间的差向异构化。我们的研究结果表明，Pel依赖性生物膜的形成需要UDP-GlcNAc C4-差向异构酶，其产生Pel合酶机器用于聚合物生产所需的UDP-GalNAc前体。
Pel is a GalNAc-rich bacterial polysaccharide that contributes to the structure and function ofPseudomonas aeruginosabiofilms. ThepelABCDEFGoperon is highly conserved among diverse bacterial species, and Pel may therefore be a widespread biofilm determinant. Previous annotation ofpelgene clusters has helped us identify an additional gene,pelX, that is present adjacent topelABCDEFGin >100 different bacterial species. ThepelXgene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have usedPseudomonas protegensPf-5 as a model to elucidate PelX function asPseudomonas aeruginosalacks apelXhomologue in itspelgene cluster. We found thatP. protegensforms Pel-dependent biofilms; however, despite expression ofpelXunder these conditions, biofilm formation was unaffected in a Delta pelXstrain. This observation led us to identify apelXparalogue, PFL_5533, which we designate here PgnE, that appears to be functionally redundant topelX. In line with this, a Delta pelX Delta pgnEdouble mutant was substantially impaired in its ability to form Pel-dependent biofilms. To understand the molecular basis for this observation, we determined the structure of PelX to 2.1 angstrom resolution. The structure revealed that PelX resembles UDP-GlcNAc C4-epimerases. Using(1)H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Our results indicate that Pel-dependent biofilm formation requires a UDP-GlcNAc C4-epimerase that generates the UDP-GalNAc precursors required by the Pel synthase machinery for polymer production.