PknB-Mediated Phosphorylation of a Novel Substrate, N-Acetylglucosamine-1-Phosphate Uridyltransferase, Modulates Its Acetyltransferase Activity
PknB-Mediated Phosphorylation of a Novel Substrate, N-Acetylglucosamine-1-Phosphate Uridyltransferase, Modulates Its Acetyltransferase Activity
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DOI：
10.1016/j.jmb.2008.12.031
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发表时间：
2009-02-20
影响因子：
5.6
通讯作者：
Nandicoori, Vinay Kumar
中科院分区：
文献类型：
--
作者：
Parikh, Amit;Verma, Sunil Kumar;Nandicoori, Vinay Kumar
确定激酶的直接靶点并确定它们的活动如何受到调控，是理解它们如何产生生物反应的核心。遗传和生化研究表明，结核分枝杆菌丝氨酸/苏氨酸蛋白激酶PKNA和PKNB在调节细胞形状和可能的细胞分裂中发挥作用。在这篇报道中，我们发现结核分枝杆菌的N-乙酰氨基葡萄糖-1-磷酸尿苷转移酶(GlmU)是一种新的底物，在苏氨酸残基上被磷酸化。GlmU具有两个重要的生化活性：C-末端结构域催化乙酰基从乙酰辅酶A转移到氨基葡萄糖-1-磷酸生成N-乙酰氨基葡萄糖-1-磷酸，N-乙酰氨基葡萄糖-1-磷酸通过尿苷5‘-单磷酸(从5’-三磷酸尿苷)转移到UDP-N-乙酰氨基葡萄糖，该反应由N-末端结构域催化。我们测定了APE形式和UDP-N-乙酰氨基葡萄糖结合形式的GlmU的晶体结构，并对其进行了分析，以确定可能与PKnB结合的苏氨酸残基。该结构显示两个结构域的结构，N-末端结构域具有类似α/β的折叠，而C-末端结构域形成左手平行的β-螺旋结构。以GlmU的N-末端结构域和C-末端结构域为底物，用PnuB进行的蛋白激酶分析表明，PnuB在C-末端区域磷酸化了GlmU。此外，突变研究显示，存在于区域414439的五个苏氨酸中的一个被pKnB磷酸化。结构和生化分析表明，可变的C末端尾巴在调节乙酰转移酶活性方面具有重要意义。值得注意的是，我们证明了尽管PnuB介导的GlmU的磷酸化不影响其尿苷转移酶的活性，但它显著地调节了乙酰基转移酶的活性。这些发现暗示PKnB通过调节GlmU的乙酰基转移酶活性来调节肽聚糖的合成。(C)2008爱思唯尔有限公司。保留所有权利。
Identifying direct targets of kinases and determining how their activities are regulated are central to understanding how they generate biological responses. Genetic and biochemical studies have shown that Mycobacterium tuberculosis serine/threonine protein kinases PknA and PknB play a role in modulating cell shape and possibly cell division. In this report, we show that the enzyme N-acetylglucosamine-1-phosphate uridyltransferase (GlmU) of M. tuberculosis is a novel substrate of PknB and is phosphorylated on threonine residues. GlmU carries out two important biochemical activities: a C-terminal domain catalyzes the transfer of acetyl group from acetyl coenzyme A to glucosamine-1-phosphate to produce N-acetylglucosamine-1-phosphate, which is converted into UDP-N-acetylglucosamine by the transfer of uridine 5'-monophosphate (from uridine 5'-triphosphate), a reaction catalyzed by the N-terminal domain. We determined the crystal structures of GlmU in ape, form and UDP-N-acetylglucosamine-bound form, and analyzed them to identify threonine residues that may be accessible to PknB. The structure shows a two-domain architecture, with an N-terminal domain having an alpha/beta-like fold and with a C-terminal domain that forms a left-handed parallel beta-helix structure. Kinase assays with PknB using the N- and C-terminal domains of GlmU as Substrates illustrated that PknB phosphorylates GlmU in the C-terminal domain. Furthermore, mutational studies reveal one of the five threonines present in region 414439 to be phosphorylated by PknB. Structural and biochemical analyses have shown the significance of a variable C-terminal tail in regulating acetyltransferase activity. Notably, we demonstrate that although PknB-mediated phosphorylation of GlmU does not affect its uridyltransferase activity, it significantly modulates the acetyltransferase activity. These findings imply a role for PknB in regulating peptidoglycan synthesis by modulating the acetyltransferase activity of GlmU. (C) 2008 Elsevier Ltd. All rights reserved.