Structural and Functional Studies of QdtC: An N-Acetyltransferase Required for the Biosynthesis of dTDP-3-Acetamido-3,6-dideoxy-α-d-glucose

Abstract

Abstract3-acetamido-3,6-didexoy-α-D-glucose or Quip3NAc is an unusual dideoxy sugar found in the Oantigens of various Gram-negative bacteria and in the S-layer glycoprotein glycans of some Grampositive bacteria. It is produced in these organisms as a dTDP-linked sugar, with five enzymes ultimately required for its biosynthesis. The focus of this investigation is on the enzyme QdtC, a CoA-dependent N-acetyltransferase that catalyzes the last step in the Quip3NAc biosynthetic pathway. For this analysis, three crystal structures were determined: the wild-type enzyme in the presence of acetyl-CoA, and two ternary complexes of the enzyme with CoA and either dTDP-DQuip3N or dTDP-3-amino-3,6-didexoy-α-D-galactose (dTDP-D-Fucp3N). Each subunit of the trimeric enzyme is dominated by a left-handed β-helix motif with 11 turns. The three active sites are located at the subunit:subunit interfaces, and the two dTDP-sugar ligands employed in this study bind to the protein in nearly identical manners. Those residues responsible for anchoring the hexose moieties of the dTDP-sugars to the protein include Glu 141, Asn 159, Asp 160 from one subunit and His 134 from another subunit. To probe the roles of various amino acid residues in the catalytic mechanism of the enzyme, ten site-directed mutant proteins were constructed and their kinetic parameters measured. On the basis of these data, a catalytic mechanism is proposed for QdtC whereby the acetylation of the sugar amino group does not require a catalytic base provided by the protein.Rather, the sulfur of CoA functions as the ultimate proton acceptor.Unusual deoxysugars are found throughout Nature, often in the lipopolysaccharides of Gramnegative bacteria (1) and on various antibiotics (1,2), antifungals (3), anthelmintics (4), and antitumor drugs (5). One such sugar derivative, 3-acetamido-3,6-dideoxy-α-D-glucose or Quip3NAc, has been observed in the O-antigens of various Gram-negative bacteria including Escherichia coli O114 (6) and in the S-layer glycoprotein glycans of some Gram-positive bacteria (7). Nucleotide-activated sugar precursors such as dTDP-D-Quip3NAc serve as the building blocks for the formation of either the S-layer glycans or the O-antigens.In Thermoanaerobacterium thermosaccharolyticum E207-71, a Gram positive, anaerobic, thermophilic organism, five enzymes are required for the biosynthesis of dTDP-D-Quip3NAc starting from glucose-1-phosphate (Scheme 1). 3,6-dideoxyhexoses, the formation of this unusual sugar begins with the attachment of α-Dglucose-1-phosphate to a nucleotide via the action of glucose-1-phosphate thymidylyltransferase (RmlA). In the next step, the 6'-hydroxyl group is removed and the C-4' hydroxyl group is oxidized to a keto-functionality yielding dTDP-4-keto-6-deoxyglucose. This reaction is catalyzed by dTDP-glucose 4,6-dehydratase (RmlB). Both the thymidylyltransferase and the dehydratase have been well characterized with respect to structure and function (8).Three additional enzymes are ultimately required for the synthesis of dTDP-D-Quip3N...

Structure of Soybean Serine Acetyltransferase and Formation of the Cysteine Regulatory Complex as a Molecular Chaperone

“…The overall fold of the GmSAT monomer is similar to the bacterial and Entamoeba homologs with a root mean square deviation of 1.0 -2.8 Å for C ␣ atoms over ϳ190 -230 residues (54 -58). Additional structural homology with a variety of acyltransferases, including QdtC required for synthesis of dTDP-3-acetamido-3,6-dideoxy-␣-D-glucose, perosamine N-acetyltransferase, N-acetylglucosamine-1-phosphate uridyltransferase, and PglD from the UDP-N,NЈ-diacetylbacillosamine synthesis pathway (65)(66)(67)(68), was detected using the DALI server and displayed 1.1-7.2 Å root mean square deviation of C ␣ atoms over ϳ90 -130 residues, largely in the left-handed ␤-helix domain. Crystallo- graphic symmetry of GmSAT generates a trimeric structure from each chain that adopts the dimer of trimers packing observed for the bacterial SAT structures (Fig.…”

“…2 and 6). The overall structure of the hexapeptide repeat that defines the left-handed ␤-helical fold of SAT and other O-acyltransferases provides a highly adaptable scaffold for tailoring of substrate specificity (65)(66)(67)(68)(69). Across the O-acyltransferase family of enzymes, oligomerization of monomers into trimeric assemblies with the length of the left-handed ␤-helical fold running along the 3-fold axis provides for substrate binding at the interfaces between monomers (65-69) (Fig.…”

“…3). In addition to their overall structural organization, the x-ray crystal structures of various O-acyltransferases show that a loop corresponding to the ␤1c-␤2c loop of GmSAT extends from the ␤-helix of one monomer to interact with a neighboring subunit to act as a cover of the active site (65)(66)(67)(68)(69). Moreover, the active sites of these enzymes share a common catalytic mechanism that relies on a histidine as the general base (65)(66)(67)(68)(69).…”

Biochemical Analysis and Structure Determination of Bacterial Acetyltransferases Responsible for the Biosynthesis of UDP-N,N′-Diacetylbacillosamine

“…In particular, WeeI binds to UDP-4-amino with a significantly lower affinity (10-fold) in comparison with PglD and PglB-ATD (Table 2). From a structural alignment standpoint, WeeI contains one key residue (Phe 13 ) that may be responsible for this dramatic K m shift. In PglD, this position (His 15 ) undergoes a conformational change to accommodate UDP-4-amino binding and interacts with sugar ␤-phosphate.…”

“…The oligomeric state of PglD consists of a homotrimer that utilizes the left-handed ␤-helix motif of two protomers to form the cleft for AcCoA binding. Structures of other bacterial N-acetyltransferases have recently been reported (13)(14)(15), although they are distant homologues of PglD based upon their divergent sugar substrates. However, the sugar acetyltransferases maintain the same overall protein fold by forming a trimer as the biological unit.…”