10 A niversary of IIMCB

A B
Fig. 2. Comparison of the DNA co-crystal structures of (A) BcnI and (B) MvaI. The figure was adapted from Sokolowska et al., Cell Mol Life Sci 2007, 64:
2351-2357.
Ecl18kI and PspGI, but cleave them with a different stagger.
Nevertheless, MvaI and BcnI have evolved a very different
strategy to deal with the asymmetries of their substrates:
both enzymes bind their substrates as monomers. As
there is only one active site per monomer, the implication
is that MvaI and BcnI must cleave the two DNA strands one
after another, with an intermittent DNA rebinding event to
bring the uncleaved strand of the nicked intermediate into
a position proximal to the active site. Support for a nicked
intermediate in the DNA cleavage reactions by MvaI and
BcnI is also provided by comparison of the MvaI and BcnI
structures with all structures in the Protein Data Bank,
because it turns out the MvaI and BcnI are more similar to
the DNA nickase MutH, a component of the mismatch repair
machinery, than to any other DNA restriction endonuclease
of known structure (Fig. 2).
The ββα-Me restriction endonuclease Hpy99I: This
restriction endonuclease is specific for the sequence
CGWCG/ and cuts DNA into fragments with highly unusual
5 nucleotide long 3’-overhangs. Our recent crystal structure
of this enzyme represents the first structure of a ββα-Me
restriction endonuclease and allows detailed comparisons
36 Annual Report 2008
with previously determined structures of ββα-Me
endonucleases that play no role in restriction biology, but are
involved in unspecific DNA degradation (such as the Serratia
nuclease), in homing (such as I-PpoI) or Holliday junction
resolution (T4 endonuclease VII). Hpy99I distinguishes
between W and S at the center of its target sequence by
exclusive minor groove readout.
Unlike major readout, minor groove readout is perfectly
suitable to distinguish S and W. The presence of an amino
group (of guanine) in the central minor groove position signals
a G:C/C:G pair, its absence (which is verified by two Hpy99I
arginines) confirms the presence of an A:T/T:A pair. Based on
mutagenesis data alone, this mechanism has been suggested
earlier for methyltransferases. To our knowledge, our Hpy99I-
DNA co-crystal structure provides its first crystallographic
demonstration.
Peptidoglycan amidases
Bacterial peptidoglycan amidases are a diverse group
of enzymes. Some are metallopeptidases, others serine
peptidases, and yet others cysteine peptidases. Only aspartic
(and threonine) peptidases have so far not been found.
In each catalytic group, different folds can be discerned,
Fig. 3. (A) Hydrogen bonding patterns of T:A/A:T pairs (W) and G:C/C:G pairs (S). The figure highlights the similarity of T:A and G:C pairs on the major
groove side, as well as the similarity of A:T and C:G pairs, which makes the distinction between W and S by major groove readout difficult. (B) Recognition
of the symmetry violating A:T/T:A pair by Hpy99I. This enzyme “verifies” the absence of a guanine amino group in the central minor groove position,
which could clash with the two arginine residues of the protein. The figure has been adapted from Szczepanowski et al., Nucleic Acids Res. 2008,
36:6109-17 and from Sokolowska et al., Nucleic Acid Research 2009, in press.