One of the main barriers to the enzymatic hydrolysis of cellulose results from its highly crystalline structure. Pretreating biomass with ionic liquids (IL) increases enzyme accessibility and cellulose recovery through precipitation with an anti-solvent. For an industrially feasible pretreatment and hydrolysis process, it is necessary to develop cellulases that are stable and active in the presence of small amounts of ILs co-precipitated with recovered cellulose. However, a significant decrease in cellulase activity in the presence of trace amounts of ILs has been reported in the literature, necessitating extensive processing to remove residual ILs from the regenerated cellulose. Towards that end, we have investigated the stability of hyperthermophilic enzymes in the presence of the IL 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]) and compared it to the industrial benchmark Trichoderma viride (T. viride) cellulase. The endoglucanase from a hyperthermophilic bacterium, Thermatoga maritima, and a hyperthermophilic archaeon, Pyrococcus horikoshii, were over expressed in E. coli and purified to homogeneity. Under their optimum conditions, both hyperthermophilic enzymes showed significantly higher [C2mim][OAc] tolerance than T. viride cellulase. Using differential scanning calorimetry we determined the effect of [C2mim][OAc] on protein stability and our data indicates that higher concentrations of IL correlated with lowered protein stability. Both hyperthermophilic enzymes were active on [C2mim][OAc] pretreated Avicel and corn stover. Furthermore, these enzymes can be recovered with little loss in activity after exposure to 15% [C2mim][OAc] for 15 h. These results demonstrate the potential of using IL-tolerant extremophilic cellulases for hydrolysis of IL-pretreated lignocellulosic biomass, for biofuel production.

Methyltransferases that employ cobalamin cofactors, or their analogues the cobamides, as intermediates in catalysis of methyl transfer play vital roles in energy generation in anaerobic unicellular organisms. In a broader range of organisms they are involved in the conversion of homocysteine to methionine. Although the individual methyl transfer reactions catalyzed are simple S N 2 displacements, the required change in coordination at the cobalt of the cobalamin or cobamide cofactors and the lability of the reduced Co +1 intermediates introduces the necessity for complex conformational changes during the catalytic cycle. Recent spectroscopic and structural studies on several of these methyltransferases have helped to reveal the strategies by which these conformational changes are facilitated and controlled.

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Cobalamin-dependent methionine synthase (MetH) is a modular protein that catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to produce methionine and tetrahydrofolate. The cobalamin cofactor, which serves as both acceptor and donor of the methyl group, is oxidized once every Ϸ2,000 catalytic cycles and must be reactivated by the uptake of an electron from reduced flavodoxin and a methyl group from Sadenosyl-L-methionine (AdoMet). Previous structures of a C-terminal fragment of MetH (MetH CT ) revealed a reactivation conformation that juxtaposes the cobalamin-and AdoMet-binding domains. Here we describe 2 structures of a disulfide stabilized MetH CT (S-SMetH CT ) that offer further insight into the reactivation of MetH. The structure of S-SMetH CT with cob(II)alamin and Sadenosyl-L-homocysteine represents the enzyme in the reactivation step preceding electron transfer from flavodoxin. The structure supports earlier suggestions that the enzyme acts to lower the reduction potential of the Co(II)/Co(I) couple by elongating the bond between the cobalt and its upper axial water ligand, effectively making the cobalt 4-coordinate, and illuminates the role of Tyr-1139 in the stabilization of this 4-coordinate state. The structure of S-SMetH CT with aquocobalamin may represent a transient state at the end of reactivation as the newly remethylated 5-coordinate methylcobalamin returns to the 6-coordinate state, triggering the rearrangement to a catalytic conformation.corrinoid methyltransferase ͉ multimodular protein ͉ protein conformation ͉ cob(II)alamin coordination ͉ enzyme catalysis C obalamin-dependent methionine synthase (MetH) is a modular enzyme (1) that catalyzes methyl transfer from methyltetrahydrofolate (CH 3 -H 4 folate) to homocysteine (Hcy), forming tetrahydrofolate (H 4 folate) and methionine. The enzyme from Escherichia coli is one of the best-studied members of a large class of cobalamin-and corrinoid-dependent methyltransferases (2). In this class of enzymes, the cobalamin cofactor plays a crucial role in catalysis, acting both as a methyl donor and acceptor. During the MetH reaction cycle the methylcobalamin form of the cofactor [MeCo(III)Cbl] is demethylated by Hcy. The electrons of the carbon-cobalt bond remain on the cofactor producing cob(I)alamin [Co(I)Cbl]. Co(I)Cbl is subsequently remethylated by CH 3 -H 4 folate regenerating the MeCo(III)Cbl cofactor (Fig. 1A). However under aerobic conditions, the cobalamin cofactor is oxidized to an inactive cob(II)alamin [Co(II)Cbl] form about once in every 2,000 turnovers (3). To avoid accumulation of this inactive species, the enzyme undergoes a reductive reactivation, in which Co(II)Cbl is reduced to Co(I)Cbl with an electron supplied by flavodoxin (Fld), and Co(I)Cbl is then remethylated using S-adenosylmethionine (AdoMet) (4, 5). Reductive reactivation is a challenging reaction because the reduction potential of Co(II)Cbl in solution is out of the range of physiological reducing agents (6-8).MetH has 4 functional units th...