Pallares Ivan G, Moore Theodore C, Escalante-Semerena Jorge C, Brunold Thomas C
Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.
Department of Microbiology, University of Georgia , Athens, Georgia 30602, United States.
Biochemistry. 2017 Jan 17;56(2):364-375. doi: 10.1021/acs.biochem.6b00750. Epub 2017 Jan 3.
The EutT enzyme from Salmonella enterica, a member of the family of ATP:cobalt(I) corrinoid adenosyltransferase (ACAT) enzymes, requires a divalent transition metal ion for catalysis, with Fe(II) yielding the highest activity. EutT contains a unique cysteine-rich HXCCXC(83) motif (where H and the last C occupy the 67th and 83rd positions, respectively, in the amino acid sequence) not found in other ACATs and employs an unprecedented mechanism for the formation of adenosylcobalamin. Recent kinetic and spectroscopic studies of this enzyme revealed that residues in the HXCCXC(83) motif are required for the tight binding of the divalent metal ion and are critical for the formation of a four-coordinate (4c) cob(II)alamin [Co(II)Cbl] intermediate in the catalytic cycle. However, it remained unknown which, if any, of the residues in the HXCCXC(83) motif bind the divalent metal ion. To address this issue, we have characterized Co(II)-substituted wild-type EutT (EutT/Co) by using electronic absorption, electron paramagnetic resonance, and magnetic circular dichroism (MCD) spectroscopies. Our results indicate that the reduced catalytic activity of EutT/Co relative to that of the Fe(II)-containing enzyme arises from the incomplete incorporation of Co(II) ions and, thus, a decrease in the relative population of 4c Co(II)Cbl. Our MCD data for EutT/Co also reveal that the Co(II) ions reside in a distorted tetrahedral coordination environment with direct cysteine sulfur ligation. Additional spectroscopic studies of EutT/Co variants possessing a single alanine substitution of either His67, His75, Cys79, Cys80, or Cys83 indicate that Cys80 coordinates to the Co(II) ion, while the additional residues are important for maintaining the structural integrity and/or high affinity of the metal binding site.
来自肠炎沙门氏菌的EutT酶是ATP：钴（I）类咕啉腺苷转移酶（ACAT）家族的成员，催化反应需要二价过渡金属离子，其中Fe（II）的活性最高。EutT含有一个独特的富含半胱氨酸的HXCCXC（83）基序（其中H和最后一个C分别位于氨基酸序列的第67位和第83位），这在其他ACAT中未发现，并且采用了前所未有的机制来形成腺苷钴胺素。最近对该酶的动力学和光谱研究表明，HXCCXC（83）基序中的残基对于二价金属离子的紧密结合是必需的，并且对于催化循环中四配位（4c）钴（II）钴胺素[Co（II）Cbl]中间体的形成至关重要。然而，尚不清楚HXCCXC（83）基序中的哪些残基（如果有的话）结合二价金属离子。为了解决这个问题，我们通过电子吸收光谱、电子顺磁共振光谱和磁圆二色性（MCD）光谱对Co（II）取代的野生型EutT（EutT/Co）进行了表征。我们的结果表明，EutT/Co相对于含Fe（II）酶的催化活性降低是由于Co（II）离子的不完全掺入，因此4c Co（II）Cbl的相对丰度降低。我们对EutT/Co的MCD数据还表明，Co（II）离子存在于扭曲的四面体配位环境中，直接与半胱氨酸硫配位。对具有His67、His75、Cys79、Cys80或Cys83单个丙氨酸取代的EutT/Co变体的额外光谱研究表明，Cys80与Co（II）离子配位，而其他残基对于维持金属结合位点的结构完整性和/或高亲和力很重要。
