Characterization of the Coenzyme‐B12–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli
Authors: Zelder Oskar , Beatrix Birgitta , Leutbecher Ulrich , Buckel Wolfgang 
Published: March 2005

The glutamate mutase dependent on adenosylcobalamin (coenzyme B12) catalyzes the carbon skeleton rearrangement of (S)‐glutamate to (2S, 3S)‐3‐methylaspartate, the first step of the glutamate fermentation pathway of the anaerobic bacterium Clostridium cochlearium. The enzyme consists of two protein components, E, a dimer ɛ2 (ɛ, 53.5 kDa) and S, a monomer (σ, 14.8 ma). The corresponding genes (glmE and glmS) were cloned, sequenced and over‐expressed in Escherichia coli. The genes glmS and glmE are separated by glmL encoding a protein of unknown function. The deduced amino acid sequence of GlmL contains an ATP‐binding motif which is common to chaperones of the HSP70–type, actin and procaryotic cell‐cycle proteins.

Both components of glutamate mutase were purified with excellent yields from cell‐free extracts of E. coli carrying the corresponding genes. In contrast to component E, component S was shown to bind coenzyme B12. This observation strongly supports the idea that significant similarities of the amino acid sequences of component S and several other cobamide‐dependent enzymes represent a common binding motif. Incubation of pure components E and S with coenzyme B12 resulted in the formation of a fully active glutamate mutase heterotetramer (ɛ2σ2) containing one molecule of coenzyme B12.

EPR spectra of recombinant glutamate mutase, now available in sufficiently large amounts, were recorded after incubation of the enzyme with coenzyme B12 and (S)‐glutamate. The EPR signals (gx,y≈ 2.1, gz= 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(I1), which is involved in the formation of the paramagnetic species but is different from cob(II)alamin (gx,y= 2.25). The spin concentration was 34–50% of the concentration of the enzyme (ɛ2σ2) coenzyme complex. The competitive inhibitors (2S, 4S)‐4‐fluoroglutamate and 2‐methyleneglutarate induced similar but not identical signals with spin concentrations of 134–148% of the enzyme concentration. Even (S)–[2,3,3,4,4–ZH], glutaate induced a signal significantly different to that of (S)‐glutamate with an intensity of only 7%. These data suggest an involvement of the Co(II)‐containing paramagnetic species in catalysis, the concentration of which reflects a steady state between its formation and decomposition. The large difference in the spin concentrations observed with (S)‐glutamate as compared to the perdeuterated glutamate is probably due to a kinetic isotope effect and indicates a cleavage of a C‐H bond during formation of the paramagnetic species. It is discussed that the paramagnetic species represents a radical pair composed of Co(II) and an organic radical.

Overnight incubation of glutamate mutase and coenzyme with substrate or one of the competitive inhibitors resulted in an inactive enzyme and cob(II)alamin, which was identified by EPR spectroscopy (gx,y= 2.25). This cob(II)alamin appeared to be very similar to a cob(II)amide present in inactive glutamate mutase preparations from C. cochlearium.