Cofactor Tail Length Modulates Catalysis of Bacterial F(420)-Dependent Oxidoreductases

F(420) is a microbial cofactor that mediates a wide range of physiologically important and industrially relevant redox reactions, including in methanogenesis and tetracycline biosynthesis. This deazaflavin comprises a redox-active isoalloxazine headgroup conjugated to a lactyloligoglutamyl tail. Here we studied the catalytic significance of the oligoglutamate chain, which differs in length between bacteria and archaea. We purified short-chain F(420) (two glutamates) from a methanogen isolate and long-chain F(420) (five to eight glutamates) from a recombinant mycobacterium, confirming their different chain lengths by HPLC and LC/MS analysis. F(420) purified from both sources was catalytically compatible with purified enzymes from the three major bacterial families of F(420)-dependent oxidoreductases. However, long-chain F(420) bound to these enzymes with a six- to ten-fold higher affinity than short-chain F(420). The cofactor side chain also significantly modulated the kinetics of the enzymes, with long-chain F(420) increasing the substrate affinity (lower K(m)) but reducing the turnover rate (lower k(cat)) of the enzymes. Molecular dynamics simulations and comparative structural analysis suggest that the oligoglutamate chain of F(420) makes dynamic electrostatic interactions with conserved surface residues of the oxidoreductases while the headgroup binds the catalytic site. In conjunction with the kinetic data, this suggests that electrostatic interactions made by the oligoglutamate tail result in higher-affinity, lower-turnover catalysis. Physiologically, we propose that bacteria have selected for long-chain F(420) to better control cellular redox reactions despite tradeoffs in catalytic rate. Conversely, this suggests that industrial use of shorter-length F(420) will greatly increase the rates of bioremediation and biocatalysis processes relying on purified F(420)-dependent oxidoreductases.