Characterization of a Structurally Distinct ATP-Dependent Reactivating Factor of Adenosylcobalamin-Dependent Lysine 5,6-Aminomutase

IF 2.9 3区 生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY Biochemistry Biochemistry Pub Date : 2024-03-12 DOI:10.1021/acs.biochem.3c00653
Amanda L. Darbyshire,  and , Kirsten R. Wolthers*, 
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Abstract

Several anaerobic bacterial species, including the Gram-negative oral bacterium Fusobacterium nucleatum, ferment lysine to produce butyrate, acetate, and ammonia. The second step of the metabolic pathway─isomerization of β-l-lysine to erythro-3,5-diaminohexanoate─is catalyzed by the adenosylcobalamin (AdoCbl) and pyridoxal 5′-phosphate (PLP)-dependent enzyme, lysine 5,6-aminomutase (5,6-LAM). Similar to other AdoCbl-dependent enzymes, 5,6-LAM undergoes mechanism-based inactivation due to loss of the AdoCbl 5′-deoxyadenosyl moiety and oxidation of the cob(II)alamin intermediate to hydroxocob(III)alamin. Herein, we identified kamB and kamC, two genes responsible for ATP-dependent reactivation of 5,6-LAM. KamB and KamC, which are encoded upstream of the genes corresponding to α and β subunits of 5,6-LAM (kamD and kamE), co-purified following coexpression of the genes in Escherichia coli. KamBC exhibited a basal level of ATP-hydrolyzing activity that was increased 35% in a reaction mixture that facilitated 5,6-LAM turnover with β-l-lysine or d,l-lysine. Ultraviolet–visible (UV–vis) spectroscopic studies performed under anaerobic conditions revealed that KamBC in the presence of ATP/Mg2+ increased the steady-state concentration of the cob(II)alamin intermediate in the presence of excess β-l-lysine. Using a coupled UV–visible spectroscopic assay, we show that KamBC is able to reactivate 5,6-LAM through exchange of the damaged hydroxocob(III)alamin for AdoCbl. KamBC is also specific for 5,6-LAM as it had no effect on the rate of substrate-induced inactivation of the homologue, ornithine 4,5-aminomutase. Based on sequence homology, KamBC is structurally distinct from previously characterized B12 chaperones and reactivases, and correspondingly adds to the list of proteins that have evolved to maintain the cellular activity of B12 enzymes.

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依赖腺苷钴胺素的赖氨酸 5,6-氨基转化酶的一种结构独特的 ATP 依赖性再活化因子的特征。
包括革兰氏阴性口腔细菌 Fusobacterium nucleatum 在内的几种厌氧细菌会发酵赖氨酸,生成丁酸盐、醋酸盐和氨。代谢途径的第二步--β-l-赖氨酸异构化为赤式-3,5-二氨基己酸--由依赖于腺苷钴胺(AdoCbl)和 5'-磷酸吡哆醛(PLP)的酶--赖氨酸 5,6-氨基转化酶(5,6-LAM)催化。与其他依赖 AdoCbl 的酶类似,5,6-LAM 也会因 AdoCbl 5'-deoxyadenosyl 分子的丧失以及 cob(II)alamin 中间体氧化为羟基ob(III)alamin 而发生基于机理的失活。在此,我们确定了 kamB 和 kamC 这两个基因,它们负责 5,6-LAM 的 ATP 依赖性再活化。KamB和KamC编码在对应于5,6-LAM的α和β亚基(kamD和kamE)的基因的上游,它们在大肠杆菌中共同表达后共同纯化。KamBC 显示出 ATP-水解活性的基础水平,在促进 5,6-LAM 与 β-l-赖氨酸或 d,l-赖氨酸转化的反应混合物中,该活性提高了 35%。在厌氧条件下进行的紫外-可见(UV-vis)光谱研究显示,在存在 ATP/Mg2+ 的情况下,KamBC 增加了过量 β-l-lysine 存在下钴(II)氨中间体的稳态浓度。通过紫外-可见光谱耦合测定,我们发现 KamBC 能够通过将受损的羟钴(III)氨酰胺交换为 AdoCbl 来重新激活 5,6-LAM 。KamBC 对 5,6-LAM 也具有特异性,因为它对同源物鸟氨酸-4,5-氨基转化酶的底物诱导失活速率没有影响。根据序列同源性,KamBC 在结构上有别于先前表征的 B12 合子和反应酶,并相应地增加了为维持 B12 酶的细胞活性而进化的蛋白质的种类。
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来源期刊
Biochemistry Biochemistry
Biochemistry Biochemistry 生物-生化与分子生物学
CiteScore
5.50
自引率
3.40%
发文量
336
审稿时长
1-2 weeks
期刊介绍: Biochemistry provides an international forum for publishing exceptional, rigorous, high-impact research across all of biological chemistry. This broad scope includes studies on the chemical, physical, mechanistic, and/or structural basis of biological or cell function, and encompasses the fields of chemical biology, synthetic biology, disease biology, cell biology, nucleic acid biology, neuroscience, structural biology, and biophysics. In addition to traditional Research Articles, Biochemistry also publishes Communications, Viewpoints, and Perspectives, as well as From the Bench articles that report new methods of particular interest to the biological chemistry community.
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