Structural Studies of AntD: An N-Acyltransferase Involved in the Biosynthesis of d-Anthrose

Abstract

The unusual dideoxy sugar d-anthrose has been identified as an important component in the endospores of infectious agents such as Bacillus anthracis and Bacillus cereus. Specifically, it is the terminal sugar on the bacterium's exosporium, and it provides a point of interaction between the spore and the host. The biosynthesis of d-anthrose involves numerous steps starting from α-d-glucose 1-phosphate. Here we present a combined structural and functional investigation of AntD from B. cereus. This enzyme plays a key role in d-anthrose biosynthesis by catalyzing the acylation of the C-4″ amino group of dTDP-4-amino-4,6-dideoxyglucose using 3-hydroxy-3-methylbutyryl-CoA as the acyl donor. For this investigation, two ternary complexes of AntD were determined to 1.8 Å resolution: one in which the protein contained bound β-hydroxybutyryl-CoA and dTDP and the second with CoA and dTDP-4-amino-4,6-dideoxyglucose. On the basis of these high-resolution structures, it was shown that the side chain of Asp 94 lies within hydrogen bonding distance of the sugar C-4″ amino group, and the side chain of Ser 84 resides near the carbonyl oxygen of β-hydroxybutyryl-CoA. To test the roles of these residues in the catalytic mechanism of AntD, various site-directed mutant proteins were prepared and subjected to kinetic and structural analyses. The D94A and D94N mutant proteins demonstrated enzymatic activity, albeit with significantly reduced catalytic efficiencies. The S84A mutant protein showed an approximate 10-fold decrease in activity. Interestingly, the S84C and S84T mutant proteins were both active but demonstrated substrate inhibition. The three-dimensional structures of all of the mutant proteins were nearly identical to that of the wild-type enzyme, indicating that the changes in their kinetic parameters were not due to major conformational changes. Taken together, these data suggest that Asp 94 is important for substrate binding, but probably does not function as an enzymatic base, and that Ser 84 most likely plays a role in the formation of an oxyanion hole.

Structural and Biochemical Characterization of a Bifunctional Ketoisomerase/N-Acetyltransferase from Shewanella denitrificans

“…17,19,20,26–28 These enzymes all belong to the left-handed β-helix superfamily. From these studies it has become clear that the sugar N -acyltransferases have evolved into two separate protein classes that differ with respect to substrate binding orientations and reaction mechanisms.…”

“…19,20,26 Class 2 enzymes, such as those from T. thermosaccharolyticum, Bordetella petrii , and Bacillus cereus , have active sites devoid of enzymatic bases, and thus their reaction mechanisms are thought to proceed through substrate-assisted catalysis. 17,27,28 …”

Catalytic Mechanism of Perosamine N-Acetyltransferase Revealed by High-Resolution X-ray Crystallographic Studies and Kinetic Analyses

“…This is in marked contrast to the reaction mechanisms of WlbB, QdtC, and AntD, which are N -acetyl or N -acyltransferases that have also been studied in our laboratory. 21-23 Both WlbB and QdtC are involved in the biosynthesis of unusual acetylated sugars found in the O -antigens of certain Gram-negative bacteria, and both function on C-3‴ hexose amino groups. AntD is involved in the production of d -anthrose, an important carbohydrate found in the endospores of Bacillus anthracis , the causative agent of anthrax.…”

Mutational Analysis of the Integral Membrane Methyltransferase Isoprenylcysteine Carboxyl Methyltransferase (ICMT) Reveals Potential Substrate Binding Sites

“…In AntD, an N-acyltransferase that is involved in the D-anthrose biosynthesis pathway in Bacillus anthracis, standard Michaelis-Menten kinetics were observed for the wild-type enzyme and a S84A mutant, whereas substrate inhibition kinetics were observed for S84T and S84C mutants (37). Crystal structures of AntD indicate that Ser-84 lines the substrate binding site and that the S84C mutant binds the substrate in an alternate conformation supporting the hypothesis that substrate inhibition arises for S84C due to 4A2N) is colored according to amino acid sequence conservation with the ICMT family members (pink, high sequence conservation; green, low sequence conservation) in stereoview.…”

“…AdoHcy is drawn as sticks and colored yellow. altered substrate binding (37). Similarly, in the human sulfotransferase SULT1A1, substrate inhibition by dopamine was observed when Phe-247 was mutated to leucine and was interpreted to mean that this substitution created space for the binding of a second dopamine molecule (35).…”