Glutamate mutase from Clostridium cochlearium: the structure of a coenzyme B12-dependent enzyme provides new mechanistic insights

Abstract

The long axial cobalt-nitrogen bond first observed in the structure of MCM appears to result from a contribution of the species without upper ligand. The tight binding of the tartrate ion conforms to the requirements of tight control of the reactive intermediates and suggests how the enzyme might use the substrate-binding energy to initiate cleavage of the cobalt-carbon bond. The cofactor does not appear to have a participating role during the radical rearrangement reaction.

A new paradigm for electrostatic catalysis of radical reactions in vitamin B 12 enzymes

“…This work explored, quantified, and elucidated the origin of the catalytic power of B 12 by using the EVB and LRA simulation methods that allow us to overcome the convergence difficulties of previous QM/MM approaches. It was found, in agreement with previous studies, that the catalytic power is associated with the interaction between the ribose and the protein [in particular, Glu-370, which is equivalent to Glu-330 in the mutase from Clostridium cochlearium (20)]. However, in contrast to previous studies, it was determined that the interaction between the active site and the reacting substrate is associated with electrostatic interactions rather than strain effects.…”

A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase

“…The ␣ 2 ␤ 2 tetramer is arranged such that there is no direct interaction between the active sites of either ␣␤ pair. The presence of a TIM barrel and a Rossmann domain in 5,6-LAM is consistent with the domain usage in other Cbldependent base-off enzymes, such as GM (19), MCM (20), and methionine synthase (31,32), but the orientation of the Rossmann domain relative to the TIM barrel is markedly different and is likely to be mechanistically relevant (discussed below). It is also interesting that both subunits play a role in binding both cofactors; AdoCbl is bound at the C terminus of the Rossmann domain of ␤ and by the accessory clamp at the edge of the ␣ subunit, whereas PLP is bound at the C terminus of the TIM domain of ␣ and by a lysine residue at the edge of the ␤ subunit (Fig.…”

“…The structure reveals that AdoCbl is bound by a Rossmann domain and that PLP, which is tethered to the B 12 -binding domain by means of its imine linkage, is bound in the putative active site, at the top of a triosephosphate isomerase (TIM) barrel domain. Thus, 5,6-LAM joins a group of three other AdoCbl-dependent radical enzymes (GM, MCM, and diol dehydratase) (19)(20)(21) and one AdoMet-dependent radical enzyme (biotin synthase) (22) in using the TIM barrel fold to sequester substrates that form free-radical intermediates. Our structure is distinctive among all PLP-dependent enzymes in that Abbreviations: AdoCbl, adenosylcobalamin or coenzyme B12; Ado, adenosyl group; Ado • , 5Ј-deoxyadenosyl radical; AdoH, 5Ј-deoxyadenosine; AdoMet, S-adenosylmethionine; Cbl, cobalamin; DMB, dimethylbenzimidazole; GM, glutamate mutase; 5,6-LAM, lysine 5,6-aminomutase; 2,3-LAM, lysine 2,3-aminomutase; MCM, methylmalonyl-coenzyme A mutase; PLP, pyridoxal 5Ј-phosphate; TIM, triosephosphate isomerase.…”

Structures of the N-terminal modules imply large domain motions during catalysis by methionine synthase

“…The isolated cobalaminbinding module consists of two domains, a four-helix bundle (the Cap domain) lying over the upper (␤) face of the cobalamin, and a Rossmann ␣͞␤ domain (the Cob domain) containing a critical histidine residue that coordinates the cobalt of cobalamin from beneath. In MetH, as in several other B 12 -dependent enzymes (9,10), the dimethylbenzimidazole nucleotide is displaced from the lower face of the cobalamin and extends into the core of the Rossmann fold.…”