Supporting Information Legends
Figure S1. Structure-based alignment of cdMGD1 and its closest structural homologues.
The C-domains (right), which bind the nucleotide sugar donor, show higher fold conservation than
the N-domains (left) that interact with very diverse acceptor substrates. The disordered region in
MGD1 corresponds to a region of high variability in other GT-B enzymes. Proteins in cartoon
representation colored red for cdMGD1, blue for Escherichia coli MurG (1NLM), light green for CalG1
(3OTH) from Micromonospora echinospora, and light blue for OleI (2IYA) from Streptomyces
antibioticus. The DALI Z scores (Holm and Sander, 1993), root mean square (rms) deviation and
sequence identity are as follows: MurG [Z score 25.4, rms 3.3 Å (over 295 Cαs); 19% sequence
identity]; CalG1 [Z score 21.7, rms 3.0 Å (over 284 Cαs); 16% sequence identity]; OleI [Z score 21.6,
rms 3.2 Å (over 280 Cαs); 13% sequence identity].
Figure S2. SAXS analysis of cdMGD1.
(A) Left: Superposition of the experimental scattering curve obtained for native cdMGD1 (black) with
the calculated scattering curves derived from the ab initio model of cdMGD1 (red). The P(r) function
is shown at the right top of the graph. Right: Corresponding representations for cdMGD1Δ192-215.
The Rg for cdMGD1 and cdMGD1Δ192-215 were determined using the Guinier approximation to be
3.25 nm and 2.78 nm, respectively. The particle maximum dimension (Dmax) derived from the P(r)
function is 107 Å for cdMGD1 and 97 Å for cdMGD1Δ192-215. Molecular masses were determined to
be 45.3 kDa for cdMGD1 and 40.7 kDa for cdMGD1Δ192-215, not far from the expected values of 45
and 42.7 kDa, respectively.
(B) Overlay of molecular envelope of the ab initio calculated model of cdMGD1 (orange) with the
crystal structure of cdMGD1 (red). The SAXS derived envelope is extended compared to the crystal
structure and we interpret the undefined volume to be occupied by the flexible region [W182-F230]
missing in the crystal structure.
(C) Top: Ab initio envelope of cdMGD1 (orange). Bottom: Overlay of the ab initio calculated models of
cdMGD1 and cdMGD1Δ192-215 (blue). The maximal dimensions of the models are indicated. The
deletion mutant is less extended than cdMGD1.
(D) Kratky representation of cdMGD1 (left) and cdMGD1Δ192-215 (right). The Kratky plots of the two
constructs have a characteristic bell-shaped appearance indicating that both samples are mostly
folded. However, the plot for cdMGD1 displays elevated scattering at higher angles suggesting that
part of the protein may be flexible (presumably the flexible region [W182-F230]). This behavior is less
pronounced in cdMGD1Δ192-215 where a part of this flexible region has been deleted.
Figure S3. Structure-based sequence alignment of the three most conserved regions in MGD1 and its
closest structural homologues.
For clarity, the -strands (yellow shading) and -helices (underlined) are indicated only for MGD1.
The proposed catalytic residue(s) are in red. Residues in bold blue in MGD1 sequence make contacts
with the nucleotide sugar donor. A catalytic dyad (H13 and E117 in CalG1) has been proposed for
CalG1, CalG3, CalG4, OleI and OleD. MGD1 and MurG have no equivalent of the acidic position at the
end of N4. A conserved glutamate (E438 in MGD1) is observed only in enzymes using a UDP-sugar,
not in those, like CalG1 and CalG3, which instead use a dTDP-sugar. The glutamate residue is shown
to contact both O2’ and O3’ of ribose. Sequences were retrieved from UniProtKB database: P17443
for E. coli MurG; Q9HW01 for Pseudomonas aeruginosa MurG; Q8KNF2, Q8KND7 and Q8KNC3 for
CalG1, CalG3 and CalG4 from M. echinopsora, respectively; Q3HTL7 and Q53685 for OleI and OleD
from S. antibioticus, respectively.
Figure S4. Activity assays of native MGD1 and the mutant cdMGD1192-215 measured in the
presence of PA or PG, both at 1.5 mol%.
Figure S5. Comparison of the active sites of MGD1 and other GT-B enzymes.
For clarity, only CalG1 ligands (TDP and calicheamicin) are represented. The proposed catalytic
histidine residue (H155 in MGD1), located in G-loop 1, is oriented differently in MGD1 compared to
MurG, CalG1, and OleI (left panel). However, a movement in the G-loop 1 is likely to occur upon
MGD1 membrane binding, which will bring the histidine base in a position similar to the
corresponding His in other GTs. To support this hypothesis, an alternative conformation of G-loop1
has been observed in a lower resolution model of cdMGD1:UDP. The superposition of the two
cdMGD1:UDP complexes shows a conformational rearrangement of G-loop 1 with formation of an
“extra half α-turn” on Nα1 and re-positioning of the histidine side-chain (right panel). A catalytic HisAsp/Glu dyad has been described in the active sites of CalG1 and OleI (Bolam et al., 2007; Chang et
al., 2011). Hydrogen bonding between Glu and His should facilitate deprotonation by His of the
acceptor OH-group (H13 and E117 in CalG1). No equivalent acidic residue is present in MurG and
MGD1. A histidine base in this position is not invariant across the GT-B fold family. Instead, an acidic
residue is used as a catalytic base, as shown for GtfA, a bacterial GT of the vancomycin class pathway
(Mulichak et al., 2003). Proteins are depicted as cartoon colored red for MGD1 (or grey for lower
resolution model in right panel), yellow for GtfA, light green for CalG1, light blue for OleI and blue for
E. coli MurG. Protein residues and ligands are in stick representation with carbon atoms colored as in
respective cartoon, oxygen in red, nitrogen in blue, phosphate in orange, sulfur in yellow and iodine
in purple. Images were prepared with Pymol.
Table S1. Summary of the enzyme activity of MGD1 mutants. Mutant enzyme activity was measured in
the presence of PG. Mutants in bold have been evaluated in the present paper. The equivalent residue in the
spinach enzyme has been mutated in mutants issued from (Botté et al., 2005).