STRUCTURE ANALYSIS OF B12 BINDING SUBUNIT OF GLUTAMATE MUTASE

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Coenzyme B12-dependent glutamate mutase catalyzes the isomerization of (S)-glutamate to (2S, 3S)-3-methylaspartate, the first step in glutamate fermentation by Clostridium tetanomorphum . To better understand the mechanism of glutamate mutase, and in particular how the apoprotein modulates the reactivity of the B12 cofactor, we have crystallized the B12-binding subunit of glutamate mutase (15 kDa), in the presence of coenzyme B12 (AdoCbl). Crystals as large as 1.2 mm x 0.8 mm x 0.7 mm have been obtained routinely, and some of them diffract to a resolution of 2.2
. All data sets have indexed as primitive tetragonal with unit cell dimensions a = b = 104.7
, c = 162.7
. The large unit cell indicates that the asymmetric unit likely contains four to six molecules. A novel approach to MAD phasing, employing the intrinsic cobalt of AdoCbl as an anomalous scatterer, was recently conducted at CHESS. Two data sets (91% complete to 2.6
) taken at energies corresponding to the peak and inflection points of cobalt were combined with data taken on a home source (1.5418
radiation, used as a remote wavelength). Four cobalt sites were identified in anomalous difference Patterson maps. Electron density maps generated from phases calculated by SOLVE have not been interpretable. However, the combination of phasing information from the cobalt experiments with isomorphous replacement phasing from a recently identified mercury derivative may prove adequate for structure determination.