The first published 3D structure for a GT2 protein was that of the catalytic domain of the spore coat polysaccharide biosynthesis protein, SpsA, from Bacillus subtilis (Charnock & Davies 1999; Tarbouriech et al. 2001). The SpsA is an a/P protein that adopts a GT-A fold. The high-resolution (1.5 A) 3D structure provides information on how nucleotide diphosphates might be bound by interacting loops in the active site pocket, via Mn2+ or Mg2+ ions. The SpsA structure points to the identity of active site amino acid residues in the pocket and to the likely catalytic mechanism within the GT2 group of glycosyl transferases (Tarbouriech et al. 2001 ).
Although the specificity of the enzyme is unknown, the crystal structures of SpsA are available both with and without bound UDP-Mn2+ or UDP-Mg2+, and it is assumed that a uridine nucleotide-monosaccharide is the substrate. The active site structure of SpsA synthase serves as a prototype for the organization of other family GT2 synthases. The 256-amino-acid protein has two domains, a nucleotide-binding domain and an acceptor-binding domain, and features a disordered loop spanning the active site. Both UDP-Mn2+ or UDP-Mg2+ bind in a deep cleft situated in the N-terminal domain (Fig. 5.8A). The hydrogen and ionic interactions of the enzyme with UDP- Mn2+ at the binding site are shown in Fig. 5.8B and Fig. 5.8C.
The sequence of the family GT2 bacterial (1,3)-P-d-glucan synthase (CrdS) (Stasinopoulos e t al. 1999) , which contains the extended DDD35QXXRW motif, has been threaded on to the SpsA structure and shows a similar folding pattern in the UDP-Glc-binding site of the catalytic region of SpsA (Karnezis et al. 2003 ).
D1 D2 D335 QXXRW
Axy AcsA TVDIFIPTYDX16DWPPDKVNVYILDDG
Axy Acs Al I WDVYVPSYNX16DWPADKLNVYILDDG
Axy BscA TVDIFVPTYNX16DWPPEKVRVHILDDG
Atu CelA TVDVFVPSYNX16DYPADRFTVWLLDDG
Ghi CelAI DYPVDKVSCYISDDG
Ath Irx3 DYPVEKISCYVSDDG
Ath Rswl DYPVDKVACYVSDDG
Asp CrdS LVDVFICTYNX16DYP- RLRVFVCDDN
Ddi DcsA DYPSENLWIGLLDDS
Rme NodC DYPG-ELRVYWDDG
Pmu HasA HYP- FEVIVTDDG
Spy HasA KVAAVIPSYNX16TYP- LSEIYIVDDG PCV HasA Spn Cap3B See Chs2
yigrvds - shakacnln yiirdqn - nhakacnln yiarptn - ehakacnln yltrern - vhakacnln yvsrekrpgyqhhkkacaen yvsrekrpgfqhhkkacamn yvsrekrpgfqhhkkacamn yvtrpdn - khakacnln ylrrrkpp - iphnkagnin
LMQTPHHFYSP -D LMQTPHHFYSP -D LLQTPHHFYSP -D LVQTPHFFVNP -D YVQFPQRFDGI -D LDQFPKWFPIE -D LDQFPKWYPIN -D WQTPQFYFNS -D FVQTPQFFSNIYPVD
dx46dcdx94dx35qrvrw dx46dcdx94dx35qrvrw dx46dcdx94dx35qrmrw dx65dadx94dx35qrsrw dx, 65dcdx21 0dx37qvlrw dx, 65dcdx, 99dx37qvlrw dx, 65dcdc21 6dx37qvlrw dx52dadx94dx35qrtrw dx90dadx255dx35qrkrw dx51 dsdx98dx35qqlrw dx46dadx99dx35qqnrw dx51 dsdx, 38dx35qqtrw dx43dsdx99dx35qqlrw dx103dmdx102dx38qrrrw
Axy, Acetobacter xylinus; Atu, Agrobacterium tumefaciens; Ghi, Gossypium hirsutum; Ath, Arabidopsis thaliana; Asp, Agrobacterium sp.; Ddi, Dictyostelium discoidium; Rme, Rhizobium meliloti; Pmu, Pastureiia muitocida; Spy, Streptococcus pyogenes; PCV, Chlorella virus; Spn, Streptococcus pneumoniae; See, Saccharomyces cerevisiae, Bsu, Bacillus subtiiis.
SwissProt accession numbers: AcsA (PI 9449), Acsll (Q59167), BscA (P21877), CelA (Q44418), CelAI (P93155), IrxB (AF088917), Rswl (AAC39334), CrdS (A5189370), DcsA (A500200), NodC(P0341), HasA (Pmu) (068389), HasA (Spy) Q54865, HasA (PCV) Q84419, Cap3B (P72520), Chs2 (PI 4180), SpsA (P39621).
Figure 5.8 Structure of a GT2 glycosyl transferase SpsA from Bacillus subtilis. (A) Surface representation of SpsA (PDB accession code 1QGS) showing an (a/p)8 fold with secondary structure elements (Charnock & Davies 1999). Four conservative aspartic acid residues (D39, D99, D158 and D191), depicted as red patches on the surface of the SpsA, represent the key structural elements and with two Mg2+ ions (grey spheres) they collectively coordinate the position of the UDP molecule (orange/red sticks) in the active site pocket. (B) A view of the active site depression, where the four aspartic amino acids shown in cpk sticks bind the UDP molecule (orange/red sticks). The black dashed lines indicate interactions that are critical for UDP binding with the four aspartic acid residues and Mg2+ ions. Panels A and B were prepared with PyMol (http://pymol.sourceforge.net/). (C) Hydrogen bonding and ionic interactions between amino acid residues of the active site of the GT2 protein SpsA from B. subtilis and the substrate analogue UDP-Mn. Amino acid sequences adjacent to the residues involved in binding are also shown and correspond to the sequences from SpsA from B. subtilis, CgeD from B. subtilis, 334-amino acid hypothetical protein from Pyrococcus horikoshii, glycosyltransferase involved in O- antigen synthesis from Vibrio cholerea, root nodulation factor NodC from Rhizobium sp., and cellulose synthase from Acetobacter xylinus (A subunit), respectively (Charnock & Davies 1999.)
A second 3D structure of a putative glycosyl transferase from Bacteroides fragilis belonging to the GT2 group of enzymes was recently deposited in the Protein Data Bank (PDB accession code 3BCV; K. Palani, D. Kumaran, S.K. Burley, S. Swaminathan, New York Structural GenomiX Research Consortium). This structure shows a spatial conservation of aspartic acid residues D42 and D94, which correspond to D39 and D99 in the SpsA structure. These acidic residues most likely participate in coordination of sugar nucleotides in the active site pocket of the glycosyl transferase from Bacteroides fragilis (Fig. 5.8 B).
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