Characterization of the Coenzyme‐B₁₂–Dependent Glutamate Mutase from Clostridium cochlearium Produced in Escherichia coli

Abstract

The glutamate mutase dependent on adenosylcobalamin (coenzyme B,J 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 E~ ( E , 53.5 kDa) and S, a monomer (a, 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 BIZ. 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 B,, resulted in the formation of a fully active glutamate mutase heterotetramer (~~a~) containing one molecule of coenzyme BIZ.EPR spectra of recombinant glutamate mutase, now available in sufficiently large amounts, were recorded after incubation of the enzyme with coenzyme B,, and (S)-glutamate. The EPR signals (gx, = 2.1, g, = 1.985) were of much better resolution than observed earlier with the clostridial enzyme. Their typical hyperfine splitting is clearly derived from Co(II), which is involved in the formation of the paramagnetic species but is different from cob(I1)alamin (gx,, = 2.25). The spin concentration was 34-50% of the concentration of the enzyme ( E~C T J 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,]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(I1)-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(I1) 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,, = 2.25). This cob(I1)alamin appeared to be very similar to a cob(I1)amide present in inactive glutamate mutase preparations...

Coordination of a histidine residue of the protein‐component S to the cobalt atom in coenzyme B12‐dependent glutamate mutase from Clostridium cochlearium

“…The enzyme from Clostridium cochlearium consists of two protein components, E (e2, 107 kDa) and S (a, 14.8 kDa). The active enzyme complex was characterized as a heterotetramer (e2a2) containing one coenzyme B~2 [2]. The glutamate mutase genes from C. cochlearium, glmE [2] and glmS [3], as well as from C. tetanomorphum, mutE [4~6] and mut S [6,7], have been cloned, sequenced and overexpressed in Escherichia coli.…”

“…The active enzyme complex was characterized as a heterotetramer (e2a2) containing one coenzyme B~2 [2]. The glutamate mutase genes from C. cochlearium, glmE [2] and glmS [3], as well as from C. tetanomorphum, mutE [4~6] and mut S [6,7], have been cloned, sequenced and overexpressed in Escherichia coli. The deduced amino acid sequences of the components S revealed significant similarities to a number of other cobamide-dependent enzymes [7,8] suggesting a cobamide-binding motif.…”

“…The deduced amino acid sequences of the components S revealed significant similarities to a number of other cobamide-dependent enzymes [7,8] suggesting a cobamide-binding motif. Component S from (7. cochlearium was shown to be indeed able to bind coenzyme Bl2 [2]. Electron paramagnetic resonance *Corresponding author.…”

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

“…C-Co bond homolysis and the transient formation of Ado • is triggered by substrate binding in the AdoCbl-dependent enzymes methylmalonyl-coenzyme A mutase (MCM) (14), glutamate mutase (GM) (15,16), and diol dehydratase (17), whereas effector binding triggers the C-Co bond homolysis in the AdoCbl-dependent ribonucleotide reductase (18). How substrates or effectors afford such enormous Co-C bond cleavage rate accelerations in AdoCbl-dependent enzymes is a question that has intrigued scientists for decades.…”

The Reaction of the Substrate Analog 2-Ketoglutarate with Adenosylcobalamin-dependent Glutamate Mutase

“…Glutamate mutase catalyzes the reversible isomerization of L-glutamate and L-threo-3-methylaspartate (1)(2)(3). It is one of a group of enzymes that use adenosylcobalamin (AdoCbl), 1 a biologically active form of vitamin B 12 , to catalyze unusual 1,2-rearrangements in which an electron-withdrawing group is interchanged with a hydrogen atom on an adjacent carbon (4 -6) The migrating group may be -OH, -NH 2 , or, as in the case of glutamate mutase, a carbon-containing fragment so that a skeletal rearrangement is effected (Fig.…”