Methyl-coenzyme M reductase (MCR), one of the most abundant enzymes on earth, catalyzes the reversible generation and oxidation of methane¹. Recent microbial incubations of samples from subsurface hydrocarbon reservoirs (e.g oil reservoirs, marine hydrocarbon seeps)^2-4 indicated that MCR homologues may mediate non-methane alkane oxidation, but their catalytic activity and structure remain largely unexplored. Here, we expressed the recombinant MCR of Ca. Syntrophoarchaeum caldarius in the methanogen Methanococcus maripaludis S0001. Furthermore, the purified enzyme possesses specific activity for butane oxidation (k_cat = 0.4 s^-1) and butyl-CoM reduction (k_cat = 1.8 s^-1), but not for other alkanes and corresponding alkyl-CoMs. We crystallized this reductase at a 2.11-angstrom resolution, revealing an overall structure and catalytic site akin to the canonical methyl-coenzyme M reductase. Site-directed mutagenesis determined that an alternative gas tunnel predicted from the enzyme`s structure was allowed the transport of butane but not other alkanes to the active site. Our results not only demonstrated that the molecular basis of substrate specificity for this Butyl-CoM Reductase but also identified a key residue controlling C4 alkane transport to active site. These findings expand our understanding of archaeal alkane metabolism, and offer a new perspective for carbon cycling in subsurface biosphere.
 

Butane, Butyl-CoM Reductase, Ananerobic oxidation; Mechanism