Structural Studies on a Glucosamine/Glucosaminide N-Acetyltransferase.
B.J.Dopkins,
P.A.Tipton,
J.B.Thoden,
H.M.Holden.
ABSTRACT
Glucosamine/glucosaminide N-acetyltransferase or GlmA catalyzes the transfer of
an acetyl group from acetyl CoA to the primary amino group of glucosamine. The
enzyme from Clostridium acetobutylicum is thought to be involved in cell wall
rescue. In addition to glucosamine, GlmA has been shown to function on di- and
trisaccharides of glucosamine as well. Here we present a structural and kinetic
analysis of the enzyme. For this investigation, eight structures were determined
to resolutions of 2.0 Å or better. The overall three-dimensional fold of GlmA
places it into the tandem GNAT superfamily. Each subunit of the dimer folds into
two distinct domains which exhibit high three-dimensional structural similarity.
Whereas both domains bind acetyl CoA, it is the C-terminal domain that is
catalytically competent. On the basis of the various structures determined in
this investigation, two amino acid residues were targeted for further study: Asp
287 and Tyr 297. Although their positions in the active site suggested that they
may play key roles in catalysis by functioning as active site bases and acids,
respectively, this was not borne out by characterization of the D287N and Y297F
variants. The kinetic properties revealed that both residues were important for
substrate binding but had no critical roles as acid/base catalysts. Kinetic
analyses also indicated that GlmA follows an ordered mechanism with acetyl CoA
binding first followed by glucosamine. The product N-acetylglucosamine is then
released prior to CoA. The investigation described herein provides significantly
new information on enzymes belonging to the tandem GNAT superfamily.
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