Structure and function of diadenylate cyclase DacM from Mycoplasma ovipneumoniae

Cyclic diadenosine monophosphate (c-di-AMP) is a second-messenger nucleotide that is produced by many bacteria. C-di-AMP can not only regulate bacterial growth, cell-wall homeostasis, ion transport and gene transcription, but can also be recognized by multiple sensor / receptor proteins in infected host cells to trigger an innate immune response. Mycoplasma ovipneumoniae causes non-progressive pneumonia in both sheep and goats. Here, we analyzed c-di-AMP signaling in M. ovipneumoniae, which is a genome-reduced obligately pathogenic bacterium. Our results demonstrate that these bacteria can produce c-di-AMP, and we could identify the diadenylate cyclase, which was named DacM. The enzyme was found to utilize both ATP and ADP to synthesize c-di-AMP, resembling CdaM from a novel family of diadenylate cyclases first found in Mycoplasma pneumoniae. Furthermore, we present the crystal structures of DacM in the apo state and substrate-bound state at 3 Å and 1.9 Å resolution, respectively. Mutation of residues Asp112, Gly113, Tyr128, Phe129, and Arg143 surrounding the active sites to Ala were lethal to DacM enzymatic activity. These structures provide valuable insights into the biochemistry of c-di-AMP, and offer a basis for the structure-based design of new drugs for animal husbandry.


Introduction 33
Mycoplasmas are distinguished from other bacteria by minute size and lack of a cell wall [1].

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They have been studied as some of the smallest free-living and self-replicating cells [2].

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AMP for the growth of several pathogenic bacteria is marked by an increased resistance to 53 antibiotics that target the cell wall. Moreover, the lack of DAC enzymes in humans makes them an 54 ideal target for the development of novel antibiotics. Currently, five types of DACs have been 55 identified, named DisA, CdaA (or DacA), CdaS, CdaM and CdaZ [19,22]. Most bacteria contain a single DAC enzyme, some contain multiple enzymes, such as Clostridium spp., which contains two 57 types of DAC (CdaA and DisA) and B. subtilis, which contains DisA, CdaA, and CdaS [13,14].

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However, the detailed molecular mechanism of c-di-AMP regulation is still unknown. Thus, more 59 structural and functional investigations of DAC enzymes are required for a better understanding of 60 c-di-AMP synthesis urgently.

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The

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In this study, we discovered c-di-AMP synthesis in M. ovipneumoniae strain Y98, and identified 72 the diadenylate cyclase, which we named DacM. This novel c-di-AMP synthase was found to only 73 contain a single transmembrane domain, indicating that it belongs to the CdaM-type. Although the 74 protein also contains conserved RHR (Arg-His-Arg) and DGA (Asp-Gly-Ala) motifs, the overall 75 sequence has low similarity with the reported c-di-AMP synthase sequences. Furthermore, we  To determine whether M. ovipneumoniae Queensland Strain Y98 can produce c-di-AMP, 83 nucleotides were extracted and analyzed using an ACQUITY UPLC I-Class system coupled with a 84 VION IMS QTOF mass spectrometer. The retention time (tr) of synthetic c-di-AMP was 5.71 min 85 under these conditions (Fig 1B). A peak with the same retention time was observed in the extract 86 of M. ovipneumoniae Y98 cells (Fig 1A).

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To identify potential enzymes involved in c-di-AMP synthesis in M. ovipneumoniae Y98, we 88 searched for potential proteins with a DAC domain using a whole-genome sequencing approach.

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We found a single protein containing a DAC domain, with conserved 'DGA' (Asp-Gly-Ala) and        136 ovipneumoniae Y98 could be determined at a resolution of 3 Å. The crystal belonged to space group I2 1 3 (S1 Table)

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The active sites between the two protomers of DacM were also a slightly different (Figs 4A and 179 4B). In the ATP-binding cavity, residue Tyr128 located in the loop connecting β3 and α4, which is 180 highly conserved among different homologues, engaged in a π-π-stacking interaction with the 181 adenine moiety of ATP, as was reported for CdaA [17]. Compared to the apo state, the side chain 182 of residue Tyr128 underwent an obvious rotation toward the adenine moiety of ATP (Fig 4C).

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Furthermore, the neighboring residue Phe129 also interacted with the adenine moiety via its amino 184 group and carbonyl group. Additionally, the highly conserved residues Asp112 and Gly113, which 185 belong to the 112 D 113 GA motif, as well as Arg143, which belongs to the 143 RHR motif, were all 186 involved in the interaction with the ribose moiety. Additionally, the Ala142 residue coordinated the 187 α-phosphate of ATP (Fig 4A).
However, in the ADP-binding cavity of protomer B, the side chain of the highly conserved 189 Tyr128 presented a similar orientation with the apo state ( Fig 4D). Therefore, the cavity was wider 190 than the ATP-binding cavity and more space was free for the ADP molecule to occupy (Fig 4B).

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The highly conserved residues Asp112 from the 112 D 113 GA motif and Arg143 from the 143 RHR motif

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The structural comparison among these proteins did not reveal major changes, which is 249 consistent with the results of the sequence alignment. As shown in Fig 6B,      282 Therefore, the substrate-bound structure we solved is thought to be an inactive state, and the two

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As previously elucidated, few major changes were seen between the apo state and substrate-286 bound state, except for two notable differences. One is the orientation of Tyr128 in protomer A, 287 which was found to rotate towards the nucleotide when binding ATP via a π-π-stacking interaction.

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The other is the loop region starting from β3 and near the N-terminal end of α4 helix in protomer B, 289 which makes it more accessible for ADP binding than in the apo state (Figs 4C and 4D). Notably,  (Figs 5A and 5B). Among these residues, Phe129 draw our 298 attention because its employ of enzymatic activity has not been reported in its homologues yet.

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Other residues, Asp112, Gly113, Tyr128 and Arg143 are conserved with homologues in enzymatic 300 activity as reported (17). By analysis combined with the structure-based sequence alignment 301 between DacM and its homologues from other species, which Phe129 is not strict conservation 302 (Fig. 6A), it may reveal a unique or more complicated substrate binding mechanism of DacM.

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It should be noted that the enzyme activity of the other DAC proteins requires mental ions, 304 such as Mg 2+ , Mn 2+ or Co 2+ . However, our present work is limited to discuss the metal ion 305 dependence in details. This question still awaits more structural and experimental investigation in 306 future. Taken together, our work provides a basis for the rational design for novel antibiotics that 307 can be used in animal husbandry. Nevertheless, additional efforts are needed to elucidate the entire 308 picture of c-di-AMP signaling and better understand the underlying mechanisms.

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The activity of diadenylate cyclase in vitro was measured by detecting the c-di-AMP 325 production. Briefly, the reaction was started by adding 2 μM △ 2-33 DacM to reaction mixture 326 containing 50 mM Tris-HCl pH 7.5, 10 mM MgCl 2 , 150 mM NaCl, and 0.1 mM ATP or 0.1 mM ADP.

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The c-di-AMP was detected by LC-MS[27] after reaction mixture incubated for 1 h at 37 °C. All 328 activity measurements were carried out in triplicate. Details are given in SI Materials and Methods.

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X-ray data collection and structure determination 338 All native datasets were collected at the Shanghai Synchrotron Radiation Facility beamline 339 BL17U and were processed using HKL2000[28] in conjunction with programs from the CCP4 340 suite [29]. The initial phases were obtained using the molecular replacement method in