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.
