Characterization of the ADP-β-d-manno-heptose biosynthetic enzymes from two pathogenic Vibrio strains,Applied Microbiology and Biotechnology

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Characterization of the ADP-β-d-manno-heptose biosynthetic enzymes from two pathogenic Vibrio strains
Applied Microbiology and Biotechnology ( IF 3.9 ) Pub Date : 2024-03-18 , DOI: 10.1007/s00253-024-13108-3
Zhaoxiang Shi ₁ , Yue Tang ₂ , Zhenyi Wang ₃ , Min Wang ₄ , Zijian Zhong _{2,

5} , Jingming Jia ₁ , Yihua Chen _{2,

5}

Affiliation

Abstract

ADP-activated β-d-manno-heptoses (ADP-β-d-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-d-glycero-β-d-manno-heptose (ADP-d,d-manno-heptose) and its C-6′′ epimer, ADP-l-glycero-β-d-manno-heptose (ADP-l,d-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-d,d-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-β-d-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-d,d-manno-heptose and the epimerase that converts it to ADP-l,d-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that β-d-mannose 1-phosphate, but not α-d-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldE_VC (from V. cholerae) and HldE_VP (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldE_VC and HldE_VP together with the E. coli–derived HldE_EC were thus carried out using β-d-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-β-d-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains.

Key points

• Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-β-d-manno-heptoses.

• HldEs from two Vibrio strains and E. coli could activate β-d-mannose 1-phosphate to ADP-β-d-mannose.

• Comparable nucleotidyltransfer efficiencies were observed in the kinetic studies of HldEs.

中文翻译：

两种致病性弧菌菌株 ADP-β-d-甘露庚糖生物合成酶的表征

抽象的

ADP 激活的β - d -甘露- 庚糖 (ADP- β - d - 甘露 -庚糖) 是革兰氏阴性细菌中脂多糖内核生物合成的前体。最近，ADP- d-甘油-β - d-甘露-庚糖（ADP- d ， d-甘露-庚糖）及其C-6′′差向异构体，ADP- l-甘油-β - d-甘露-庚糖（ADP-d，d-甘露-庚糖） -l ， d-甘露糖-庚糖）被鉴定为有效的病原体相关分子模式（PAMP），可以触发强大的先天免疫反应。尽管已经在几种不同的致病性革兰氏阴性细菌中研究了 ADP- d 、 d-甘露-庚糖的产生，但目前对弧菌菌株中 ADP- β - d-甘露-庚糖生物合成的了解仍然有限。在这里，我们表征了霍乱弧菌（大流行性霍乱的病原体）和副溶血性弧菌（非溶血性弧菌）的 ADP -d 、 d-甘露-庚糖生物合成酶和将其转化为 ADP- l 、 d-甘露-庚糖的差向异构酶。引起弧菌病并具有胃肠炎和伤口感染临床表现的霍乱病原体）与其来自大肠杆菌的同工酶的比较。此外，我们发现β - d-甘露糖1-磷酸，而不是α - d-甘露糖1-磷酸，可以被双功能激酶/核苷酸转移酶HldE _VC （来自霍乱弧菌）的核苷酸转移酶结构域激活为其ADP形式，并且HldE _VP （来自副溶血弧菌）。因此，使用β - d-甘露糖 1-磷酸作为模拟糖底物，对 HldE _VC和 HldE _VP的核苷酸转移酶结构域以及大肠杆菌衍生的 HldE _EC进行了动力学分析。总的来说，我们的工作表明霍乱弧菌和副溶血弧菌能够合成ADP- β - d-甘露庚糖，为进一步探索弧菌菌株甘露庚糖代谢的生理功能奠定了基础。