Suppr超能文献

古菌 GTP:AdoCbi-P 鸟苷酰转移酶(CobY)的结构与突变分析:对 GTP 结合和二聚化的深入了解。

Structure and mutational analysis of the archaeal GTP:AdoCbi-P guanylyltransferase (CobY) from Methanocaldococcus jannaschii: insights into GTP binding and dimerization.

机构信息

Department of Biochemistry, University of Wisconsin , Madison, WI 53706, USA.

出版信息

Biochemistry. 2011 Jun 14;50(23):5301-13. doi: 10.1021/bi200329t. Epub 2011 May 18.

Abstract

In archaea and bacteria, the late steps in adenosylcobalamin (AdoCbl) biosynthesis are collectively known as the nucleotide loop assembly (NLA) pathway. In the archaeal and bacterial NLA pathways, two different guanylyltransferases catalyze the activation of the corrinoid. Structural and functional studies of the bifunctional bacterial guanylyltransferase that catalyze both ATP-dependent corrinoid phosphorylation and GTP-dependent guanylylation are available, but similar studies of the monofunctional archaeal enzyme that catalyzes only GTP-dependent guanylylation are not. Herein, the three-dimensional crystal structure of the guanylyltransferase (CobY) enzyme from the archaeon Methanocaldococcus jannaschii (MjCobY) in complex with GTP is reported. The model identifies the location of the active site. An extensive mutational analysis was performed, and the functionality of the variant proteins was assessed in vivo and in vitro. Substitutions of residues Gly8, Gly153, or Asn177 resulted in ≥94% loss of catalytic activity; thus, variant proteins failed to support AdoCbl synthesis in vivo. Results from isothermal titration calorimetry experiments showed that MjCobY(G153D) had 10-fold higher affinity for GTP than MjCobY(WT) but failed to bind the corrinoid substrate. Results from Western blot analyses suggested that the above-mentioned substitutions render the protein unstable and prone to degradation; possible explanations for the observed instability of the variants are discussed within the framework of the three-dimensional crystal structure of MjCobY(G153D) in complex with GTP. The fold of MjCobY is strikingly similar to that of the N-terminal domain of Mycobacterium tuberculosis GlmU (MtbGlmU), a bifunctional acetyltransferase/uridyltransferase that catalyzes the formation of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc).

摘要

在古菌和细菌中,腺苷钴胺素 (AdoCbl) 生物合成的后期步骤统称为核苷酸环组装 (NLA) 途径。在古菌和细菌的 NLA 途径中,两种不同的鸟苷酰转移酶催化钴胺素的激活。具有催化 ATP 依赖性钴胺素磷酸化和 GTP 依赖性鸟苷酰化双重功能的细菌鸟苷酰转移酶的结构和功能研究已经完成,但仅催化 GTP 依赖性鸟苷酰化的单功能古菌酶的类似研究尚未完成。本文报道了来自古菌 Methanocaldococcus jannaschii (MjCobY) 的鸟苷酰转移酶 (CobY) 酶与 GTP 形成复合物的三维晶体结构。该模型确定了活性位点的位置。进行了广泛的突变分析,并在体内和体外评估了变体蛋白的功能。甘氨酸 8、甘氨酸 153 或天冬酰胺 177 残基的取代导致催化活性丧失≥94%;因此,变体蛋白未能在体内支持 AdoCbl 合成。等温滴定量热实验的结果表明,MjCobY(G153D) 对 GTP 的亲和力比 MjCobY(WT) 高 10 倍,但未能结合钴胺素底物。Western blot 分析的结果表明,上述取代使蛋白质不稳定且易于降解;讨论了在与 GTP 形成复合物的 MjCobY(G153D) 的三维晶体结构框架内观察到的变体不稳定性的可能解释。MjCobY 的折叠与分枝杆菌结核分枝杆菌 GlmU(MtbGlmU)的 N 端结构域非常相似,MtbGlmU 是一种具有乙酰基转移酶/尿苷酰转移酶双重功能的酶,催化尿苷二磷酸-N-乙酰葡萄糖胺 (UDP-GlcNAc) 的形成。

相似文献

3
Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii.
PLoS One. 2015 Oct 29;10(10):e0141297. doi: 10.1371/journal.pone.0141297. eCollection 2015.
5
Characterization of an Fe(2+)-dependent archaeal-specific GTP cyclohydrolase, MptA, from Methanocaldococcus jannaschii.
Biochemistry. 2007 Jun 5;46(22):6658-67. doi: 10.1021/bi700052a. Epub 2007 May 12.
10
The genome of Rhodobacter sphaeroides strain 2.4.1 encodes functional cobinamide salvaging systems of archaeal and bacterial origins.
Mol Microbiol. 2008 Nov;70(4):824-36. doi: 10.1111/j.1365-2958.2008.06437.x. Epub 2008 Sep 18.

引用本文的文献

1
Computational Strategies to Enhance Vitamin B Biosynthesis Potential of Microbes.
Curr Microbiol. 2025 Jul 4;82(8):362. doi: 10.1007/s00284-025-04325-8.
2
Corrinoid salvaging and cobamide remodeling in bacteria and archaea.
J Bacteriol. 2024 Nov 21;206(11):e0028624. doi: 10.1128/jb.00286-24. Epub 2024 Oct 15.
4
Microbial production of vitamin B: a review and future perspectives.
Microb Cell Fact. 2017 Jan 30;16(1):15. doi: 10.1186/s12934-017-0631-y.
5
Solution Structural Studies of GTP:Adenosylcobinamide-Phosphateguanylyl Transferase (CobY) from Methanocaldococcus jannaschii.
PLoS One. 2015 Oct 29;10(10):e0141297. doi: 10.1371/journal.pone.0141297. eCollection 2015.

本文引用的文献

1
Processing of X-ray diffraction data collected in oscillation mode.
Methods Enzymol. 1997;276:307-26. doi: 10.1016/S0076-6879(97)76066-X.
2
Crystal structures of ethanolamine ammonia-lyase complexed with coenzyme B12 analogs and substrates.
J Biol Chem. 2010 Aug 20;285(34):26484-93. doi: 10.1074/jbc.M110.125112. Epub 2010 Jun 1.
5
In vivo analysis of cobinamide salvaging in Rhodobacter sphaeroides strain 2.4.1.
J Bacteriol. 2009 Jun;191(12):3842-51. doi: 10.1128/JB.00230-09. Epub 2009 Apr 17.
6
Structure and function of GlmU from Mycobacterium tuberculosis.
Acta Crystallogr D Biol Crystallogr. 2009 Mar;65(Pt 3):275-83. doi: 10.1107/S0907444909001036. Epub 2009 Feb 20.
8
Protein-protein interaction and quaternary structure.
Q Rev Biophys. 2008 May;41(2):133-80. doi: 10.1017/S0033583508004708.
10
The thiamine kinase (YcfN) enzyme plays a minor but significant role in cobinamide salvaging in Salmonella enterica.
J Bacteriol. 2007 Oct;189(20):7310-5. doi: 10.1128/JB.00822-07. Epub 2007 Aug 10.

文献AI研究员

20分钟写一篇综述,助力文献阅读效率提升50倍。

立即体验

用中文搜PubMed

大模型驱动的PubMed中文搜索引擎

马上搜索

文档翻译

学术文献翻译模型,支持多种主流文档格式。

立即体验