Diguanylate cyclases (DGCs) synthesize the bacterial second messenger cyclic 3',5'-diguanosine monophosphate (
c-di-GMP), which is degraded by specific
phosphodiesterases.
c-di-GMP levels control the transition of bacteria from a motile to a biofilm-forming lifestyle. These bacterial communities are highly resistant to
antibiotic treatment and represent the predominant lifestyle in most
chronic infections. Hence, DGCs serve as starting point for the development of novel
therapeutics interfering with the second messenger-signaling network in bacteria. In previous studies, we showed that 2'(3')-O-(N-methylanthraniloyl) (MANT)- and 2',3'-O-(2,4,6-trinitrophenyl) (TNP)-substituted
nucleotides are potent adenylyl and
guanylyl cyclase inhibitors. The catalytic domain of DGCs is homologous to the mammalian
adenylyl cyclase catalytic domain. Therefore, we investigated the interaction of various MANT
purine and
pyrimidine nucleotides with the model DGC YdeH from Escherichia coli. We observed strong fluorescence resonance energy transfer between
tryptophan and
tyrosine residues of YdeH and the MANT group of MANT-NTPs (
MANT-ATP, -
CTP, -
GTP, -
ITP, -
UTP, and -
XTP) and an enhanced direct MANT fluorescence upon interaction with YdeH. We assessed the affinity of MANT-NTPs to YdeH by performing competition assays with NTPs. We conducted an
amino acid alignment of YdeH with the earlier crystallized Caulobacter crescentus DGC
PleD and found high similarities in the
nucleotide-binding site of
PleD. In vitro mass-spectrometric activity assays with YdeH resulted in the identification of new MANT/TNP
nucleotide-based inhibitors of DGC activity. Together, the analysis of interactions between MANT/TNP
nucleotides and YdeH provides a new basis for the identification and development of DGC inhibitors and allows insights into
nucleotide-
protein interactions.