Baier Florian, Chen John, Solomonson Matthew, Strynadka Natalie C J, Tokuriki Nobuhiko
†Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia, Canada.
‡Center for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada.
ACS Chem Biol. 2015 Jul 17;10(7):1684-93. doi: 10.1021/acschembio.5b00068. Epub 2015 Apr 16.
Within a superfamily, functionally diverged metalloenzymes often favor different metals as cofactors for catalysis. One hypothesis is that incorporation of alternative metals expands the catalytic repertoire of metalloenzymes and provides evolutionary springboards toward new catalytic functions. However, there is little experimental evidence that incorporation of alternative metals changes the activity profile of metalloenzymes. Here, we systematically investigate how metals alter the activity profiles of five functionally diverged enzymes of the metallo-β-lactamase (MBL) superfamily. Each enzyme was reconstituted in vitro with six different metals, Cd(2+), Co(2+), Fe(2+), Mn(2+), Ni(2+), and Zn(2+), and assayed against eight catalytically distinct hydrolytic reactions (representing native functions of MBL enzymes). We reveal that each enzyme metal isoform has a significantly different activity level for native and promiscuous reactions. Moreover, metal preferences for native versus promiscuous activities are not correlated and, in some cases, are mutually exclusive; only particular metal isoforms disclose cryptic promiscuous activities but often at the expense of the native activity. For example, the L1 B3 β-lactamase displays a 1000-fold catalytic preference for Zn(2+) over Ni(2+) for its native activity but exhibits promiscuous thioester, phosphodiester, phosphotriester, and lactonase activity only with Ni(2+). Furthermore, we find that the five MBL enzymes exist as an ensemble of various metal isoforms in vivo, and this heterogeneity results in an expanded activity profile compared to a single metal isoform. Our study suggests that promiscuous activities of metalloenzymes can stem from an ensemble of metal isoforms in the cell, which could facilitate the functional divergence of metalloenzymes.
在一个超家族中，功能上发生分化的金属酶通常倾向于选择不同的金属作为催化辅因子。一种假说认为，掺入替代金属可扩展金属酶的催化功能范围，并为新的催化功能提供进化跳板。然而，几乎没有实验证据表明掺入替代金属会改变金属酶的活性谱。在此，我们系统地研究了金属如何改变金属β-内酰胺酶（MBL）超家族中五种功能分化酶的活性谱。每种酶在体外分别用六种不同的金属Cd(2+)、Co(2+)、Fe(2+)、Mn(2+)、Ni(2+)和Zn(2+)进行重组，并针对八种催化性质不同的水解反应（代表MBL酶的天然功能）进行检测。我们发现，每种酶的金属异构体对天然反应和混杂反应具有显著不同的活性水平。此外，天然活性与混杂活性的金属偏好性不相关，在某些情况下甚至相互排斥；只有特定的金属异构体表现出隐秘的混杂活性，但往往以牺牲天然活性为代价。例如，L1 B3β-内酰胺酶对其天然活性而言，Zn(2+)的催化偏好性比Ni(2+)高1000倍，但仅与Ni(2+)一起时才表现出混杂的硫酯酶、磷酸二酯酶、磷酸三酯酶和内酯酶活性。此外，我们发现这五种MBL酶在体内以各种金属异构体的集合形式存在，与单一金属异构体相比，这种异质性导致了更广泛的活性谱。我们的研究表明，金属酶的混杂活性可能源于细胞中金属异构体的集合，这可能促进金属酶的功能分化。
