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The structure of Pseudomonas fluorescens lipoamide dehydrogenase, a dimeric
flavoenzyme with a molecular mass of 106,000 daltons, was solved by the
molecular replacement method and refined to an R-factor of 19.4% at 2.8 A
resolution. The root-mean-square difference from ideal values for bonds and
angles is 0.019 A and 3.8 degrees, respectively. The structure is closely
related to that of the same flavoprotein from Azotobacter vinelandii. The
root-mean-square difference for 932 C alpha atoms is 0.64 A, with 84% sequence
identity. The residues in the active site are identical, while 89% of the
interface residues are the same in the two enzymes. A few structural variations
provide the basis for the differences in thermostability and redox properties
between the two homologous proteins. Particularly, in the A. vinelandii molecule
a threonine to alanine (T452A) mutation leaves a buried carbonyl oxygen, located
at the subunit interface and in proximity of the flavin ring, unpaired to any
H-bond donor, probably providing an explanation for the lower stability of the
A. vinelandii enzyme with respect to the P. fluorescens enzyme. Six surface
loops, which previously could not be accurately positioned in the A. vinelandii
structure, are well defined in P. fluorescens lipoamide dehydrogenase. On the
basis of the P. fluorescens structure, the six loops could be correctly defined
also in the A. vinelandii enzyme. This is an unusual case where similar
refinement methodologies applied to two crystal forms of closely related
proteins led to electron density maps of substantially different quality. The
correct definition of these surface residues is likely to be an essential step
for revealing the structural basis of the interactions between lipoamide
dehydrogenase and the other members of the pyruvate dehydrogenase multienzyme
complex.
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