Abstract
A novel lactonase from
Mycoplasma
synoviae
53
(MS53_0025) and
Mycoplasma agalactiae
PG2 (MAG_6390)
was characterized by protein structure determination, molecular docking,
gene context analysis, and library screening. The crystal structure
of MS53_0025 was determined to a resolution of 2.06 Å. This protein
adopts a typical amidohydrolase (β/α)
8
-fold
and contains a binuclear zinc center located at the C-terminal end
of the β-barrel. A phosphate molecule was bound in the active
site and hydrogen bonds to Lys217, Lys244, Tyr245, Arg275, and Tyr278.
Both docking and gene context analysis were used to narrow the theoretical
substrate profile of the enzyme, thus directing empirical screening
to identify that MS53_0025 and MAG_6390 catalyze the hydrolysis of
d
-xylono-1,4-lactone-5-phosphate (
2
) with
k
cat
/
K
m
values of
4.7 × 10
4
and 5.7 × 10
4
M
–1
s
–1
and
l
-arabino-1,4-lactone-5-phosphate
(
7
) with
k
cat
/
K
m
values of 1.3 × 10
4
and 2.2 × 10
4
M
–1
s
–1
, respectively.
The identification of the substrate profile of these two phospho-furanose
lactonases emerged only when all methods were integrated and therefore
provides a blueprint for future substrate identification of highly
related amidohydrolase superfamily members.