Functional Studies of α-Riboside Activation by the α-Ribazole Kinase (CblS) from Geobacillus kaustophilus.
Functional Studies of α-Riboside Activation by the α-Ribazole Kinase (CblS) from Geobacillus kaustophilus.
复制标题
DOI：
10.1021/acs.biochem.1c00119
复制
发表时间：
2021-06-29
期刊：
影响因子：
2.9
通讯作者：
Escalante-Semerena JC
中科院分区：
文献类型：
--
作者：
Mattes TA;Malalasekara L;Escalante-Semerena JC
我们报道了嗜酸地质杆菌α-利巴唑(α-R)激酶(GkCblS)的初步性质，该酶在合成钴酰胺的过程中将α-R转化为α-R-磷酸(α-RP)。我们实施了一个连续的分光光度分析来获得几种潜在底物的动力学参数，并研究了该酶对α-N连接的核苷的特异性。α-R和ATP的表观Km值分别为358和297μM。我们还报道了合成和定量非商品化的α-核苷和β-利巴唑(β-R)的方法。纯化的GkCblS可激活α-R和其他α-核苷，包括α-腺苷(α-ADO)。GKCblS不能磷酸化β-N连接的糖苷，如β-腺苷(β-ADO)或β-利巴唑(β-R)。嗜草杆菌cblS+在肠道沙门氏菌亚种中的表达ENTERICA SV TyphimuriumLT2(以下简称S.Enterica)菌株缺乏烟酸盐单核苷酸：5，6-二甲基苯并咪唑磷酸核糖基转移酶(COBT)，导致多种苯并咪唑α-核糖苷被激活，苯并咪唑-钴酰胺的合成达到支持强劲生长的水平。值得注意的是，α-ADO不支持在类似条件下的生长，尽管GkCblS在体外磷酸化了α-ADO。当α-ADO浓度很高时，观察到细胞的生长。这一结果表明，在肠杆菌中，α-ADO的转运可能是低效的。我们的结论是，GkCblS对α-N-糖苷键具有特异性，但对α-核糖苷中的碱基没有特异性。
We report the initial characterization of the alpha-ribazole (α-R) kinase enzyme of Geobacillus kaustophilus (GkCblS), which converts α-R to α-R-phosphate (α-RP) during the synthesis of cobamides. We implemented a continuous spectrophotometric assay to obtain kinetic parameters for several potential substrates and to study the specificity of the enzyme for α-N-linked ribosides. The apparent Km values for α-R and ATP were 358 and 297 μM, respectively. We also report methods for synthesizing and quantifying non-commercially available α-ribosides and β-ribazole (β-R). Purified GkCblS activated α-R and other α-ribosides, including α-adenosine (α-Ado). GkCblS did not phosphorylate β-N-linked glycosides like β-adenosine (β-Ado) or β-ribazole (β-R). Expression of G. kaustophilus cblS+ in a Salmonella enterica subsp. enterica sv Typhimurium LT2 (hereafter S. enterica) strain lacking the nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole (DMB) phosphoribosyl transferase (CobT) enzyme resulted in the activation of various benzimidazole α-ribosides, and the synthesis of benzimidazolyl-cobamides to levels that supported robust growth. Notably, α-Ado did not support growth under similar conditions, in spite of the fact that GkCblS phosphorylated α-Ado in vitro. When α-Ado was provided at a very high concentration, growth was observed. This result that suggested that in S. enterica α-Ado transport may be inefficient. We conclude that GkCblS has specificity for α-N-glycosidic bonds, but not for the base in α-ribosides.
登录
查看更多内容
影响因子：
3.2
作者：
BERKOWIT.D;HUSHON, JM;AMES, BN
通讯作者：
AMES, BN
影响因子：
4.1
作者：
Valero, E;Varón, R;García-Carmona, F
通讯作者：
García-Carmona, F
影响因子：
4.2
作者：
Torres AC;Vannini V;Bonacina J;Font G;Saavedra L;Taranto MP
通讯作者：
Taranto MP
DOI：
10.1016/j.bbagen.2013.09.038
发表时间：
2014-01-01
影响因子：
3
作者：
Chan, Chi Ho;Newmister, Sean A.;Escalante-Semerena, Jorge C.
通讯作者：
Escalante-Semerena, Jorge C.
影响因子：
--
作者：
Hazra, Amrita B.;Tran, Jennifer L. A.;Taga, Michiko E.
通讯作者：
Taga, Michiko E.