The cyclic dinucleotide c-di-GMP synthesized by the diadenylate cyclase domain was recently discovered as a messenger molecule for signaling DNA breaks in Bacillus subtilis. By searching bacterial genomes, we identified a family of DHH/ DHHA1 domain proteins (COG3387) that co-occur with a subset of the diadenylate cyclase domain proteins. Here we report that the B. subtilis protein YybT, a member of the COG3387 family proteins, exhibits phosphodiesterase activity toward cyclic dinucleotides. The DHH/DHHA1 domain hydrolyzes c-di-AMP and c-di-GMP to generate the linear dinucleotides 5-pApA and 5-pGpG. The data suggest that c-di-AMP could be the physiological substrate for YybT given the physiologically relevant Michaelis-Menten constant (K m ) and the presence of YybT family proteins in the bacteria lacking c-di-GMP signaling network. The bacterial regulator ppGpp was found to be a strong competitive inhibitor of the DHH/DHHA1 domain, suggesting that YybT is under tight control during stringent response. In addition, the atypical GGDEF domain of YybT exhibits unexpected ATPase activity, distinct from the common diguanylate cyclase activity for GGDEF domains. We further demonstrate the participation of YybT in DNA damage and acid resistance by characterizing the phenotypes of the ⌬yybT mutant. The novel enzymatic activity and stress resistance together point toward a role for YybT in stress signaling and response.The cyclic dinucleotide c-di-GMP 2 has been firmly established as a major bacterial messenger molecule in recent years, with the cellular level of c-di-GMP regulated by diguanylate cyclase and phosphodiesterase domain proteins (1-3). In contrast, the existence of the structurally similar 3Ј,5Ј-cyclic diguanylate (c-di-AMP) in living cells was unknown until the recent serendipitous discovery of the dinucleotide bound by the DisA protein from Bacillus subtilis (4,5). It was found that c-di-AMP was synthesized by the diadenylate cyclase (DAC) domain of DisA via condensation of two ATP molecules. Witte et al. (5) suggested that c-di-AMP is involved in signaling DNA damage considering that the DNA integrity scanning protein DisA scouts the chromosome for DNA double-stranded breaks. Subsequent genomic mining revealed that the DAC domain proteins are widespread in bacteria and archaea, with many of them associated with putative sensor domains (6). The wide occurrence and domain architecture of the DAC domain proteins hinted that c-di-AMP may be another hidden nucleotide messenger mediating various cellular functions and phenotypes.Currently, there is no report of c-di-AMP degrading or exporting proteins for controlling cellular c-di-AMP level. To identify potential c-di-AMP degrading proteins, we searched bacterial genomes for phosphodiesterase or phosphoesterase proteins that co-occur with the DAC domain-containing proteins (6). We found that a group of multidomain proteins seems to co-occur with a subset of the DAC domain proteins, which include the homologs of YojJ and YbbP from B. subtilis. This group of pr...

EAL domain proteins are the major phosphodiesterases for maintaining the cellular concentration of second-messenger cyclic di-GMP in bacteria. Given the pivotal roles of EAL domains in the regulation of many bacterial behaviors, the elucidation of their catalytic and regulatory mechanisms would contribute to the effort of deciphering the cyclic di-GMP signaling network. Here, we present data to show that RocR, an EAL domain protein that regulates the expression of virulence genes and biofilm formation in Pseudomonas aeruginosa PAO-1, catalyzes the hydrolysis of cyclic di-GMP by using a general base-catalyzed mechanism with the assistance of Mg 2؉ ion. In addition to the five essential residues involved in Mg 2؉ binding, we propose that the essential residue E 352 functions as a general base catalyst assisting the deprotonation of Mg 2؉ -coordinated water to generate the nucleophilic hydroxide ion. The mutation of other conserved residues caused various degree of changes in the k cat or K m , leading us to propose their roles in residue positioning and substrate binding. With functions assigned to the conserved groups in the active site, we discuss the molecular basis for the lack of activity of some characterized EAL domain proteins and the possibility of predicting the phosphodiesterase activities for the vast number of EAL domains in bacterial genomes in light of the catalytic mechanism.

Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices.

Temperature-dependent hydrogen-deuterium (H͞D) exchange of the thermophilic alcohol dehydrogenase (htADH) has been studied by using liquid chromatography-coupled mass spectrometry. Analysis of the changes in H͞D exchange patterns for the proteinderived peptides suggests that some regions of htADH are in a rigid conformational substate at reduced temperatures with limited cooperative protein motion. The enzyme undergoes two discrete transitions at Ϸ30 and 45°C to attain a more dynamic conformational substate. Four of the five peptides exhibiting the transition above 40°C are in direct contact with the cofactor, and the NAD ؉ -binding affinity is also altered in this temperature range, implicating a change in the mobility of the cofactor-binding domain >45°C. By contrast, the five peptides exhibiting the transition at 30°C reside in the substrate-binding domain. This transition coincides with a change in the activation energy of k cat for hydride transfer, leading to a linear correlation between k cat and the weighted average exchange rate constant k HX(WA) for the five peptides. These observations indicate a direct coupling between hydride transfer and protein mobility in htADH, and that an increased mobility is at least partially responsible for the reduced E act at high temperature. The data provide support for the hypothesis that protein dynamics play a key role in controlling hydrogen tunneling at enzyme active sites. There has been increasing speculation that protein dynamics may play an active role in facilitating catalysis in enzymes. NMR studies of a number of enzymes such as HIV protease (1), dihydrofolate reductase (2), cyclophilin A (3), ribonuclease A (4), and ribonuclease binase (5) show flexible regions of enzyme that may be correlated with catalysis. However, the demonstration of a causal relationship between the observed protein motions and the rate of the elementary step of an enzymecatalyzed process remains a formidable experimental challenge. Due to the multidimensional and quantum-tunneling nature of hydrogen transfer reactions, the rate of hydrogen transfer is highly sensitive to protein motions that modulate the height and width of the reaction barrier (6-8). Therefore, enzymes catalyzing proton, hydride, or hydrogen atom transfer reaction are particularly valuable for understanding the role of protein motion in catalysis.Recently, experimental studies of some enzyme systems that include alcohol dehydrogenase (9), soybean lipoxygenase (10), dihydrofolate reductase (11), methylamine dehydrogenase (12), and morphinone reductase (7) have produced anomalous kinetic data that can be rationalized only by an environmentally coupled tunneling model in which heavy atom motion controls the probability of hydrogen transfer. Among these intriguing systems is the thermophilic alcohol dehydrogenase (htADH) isolated from Bacillus stearothermophilus strain LLD-R. htADH catalyzes the reversible hydride transfer between the cofactor NAD ϩ ͞NADH and the substrate alcohol͞aldehyde with different activation ener...

EAL domain-based cyclic di-GMP (c-di-GMP)-specific phosphodiesterases play important roles in bacteria by regulating the cellular concentration of the dinucleotide messenger c-di-GMP. EAL domains belong to a family of (␤/␣) 8 barrel fold enzymes that contain a functional active site loop (loop 6) for substrate binding and catalysis. By examining the two EAL domain-containing proteins RocR and PA2567 from Pseudomonas aeruginosa, we found that the catalytic activity of the EAL domains was significantly altered by mutations in the loop 6 region. The impact of the mutations ranges from apparent substrate inhibition to alteration of oligomeric structure. Moreover, we found that the catalytic activity of RocR was affected by mutating the putative phosphorylation site (D56N) in the phosphoreceiver domain, with the mutant exhibiting a significantly smaller Michealis constant (K m ) than that of the wild-type RocR. Hydrogen-deuterium exchange by mass spectrometry revealed that the decrease in K m correlates with a change of solvent accessibility in the loop 6 region. We further examined Acetobacter xylinus diguanylate cyclase 2, which is one of the proteins that contains a catalytically incompetent EAL domain with a highly degenerate loop 6. We demonstrated that the catalytic activity of the stand-alone EAL domain toward c-di-GMP could be recovered by restoring loop 6. On the basis of these observations and in conjunction with the structural data of two EAL domains, we proposed that loop 6 not only mediates the dimerization of EAL domain but also controls c-di-GMP and Mg 2؉ ion binding. Importantly, sequence analysis of the 5,862 EAL domains in the bacterial genomes revealed that about half of the EAL domains harbor a degenerate loop 6, indicating that the mutations in loop 6 may represent a divergence of function for EAL domains during evolution.

Horse myoglobin (Mb) provides a convenient "workbench" for probing the effects of electrostatics on binding and reactivity in the dynamic [Mb, cytochrome b(5)] electron-transfer (ET) complex. We have combined mutagenesis and heme neutralization to prepare a suite of six Mb surface-charge variants: the [S92D]Mb and [V67R]Mb mutants introduce additional charges on the "front" face, and incorporation of the heme di-ester into each of these neutralizes the charge on the heme propionates which further increases the positive charge on the "front" face. For this set of mutants, the nominal charge of Mb changes by -1 to +3 units relative to that for native Mb. For each member of this set, we have measured the bimolecular quenching rate constant (k(2)) for the photoinitiated (3)ZnDMb --> Fe(3+)b(5) ET reaction as a function of ionic strength. We find: (i) a dramatic decoupling of binding and reactivity, in which k(2) varies approximately 10(3)-fold within the suite of Mbs without a significant change in binding affinity; (ii) the ET reaction occurs within the "thermodynamic" or "rapid exchange" limit of the "Dynamic Docking" model, in which a large ensemble of weakly bound protein-protein configurations contribute to binding, but only a few are reactive, as shown by the fact that the zero-ionic-strength bimolecular rate constant varies exponentially with the net charge on Mb; (iii) Brownian dynamic docking profiles allow us to visualize the microscopic basis of dynamic docking. To describe these results we present a new theoretical approach which mathematically combines PATHWAY donor/acceptor coupling calculations with Poisson-Boltzmann-based electrostatics estimates of the docking energetics in a Monte Carlo (MC) sampling framework that is thus specially tailored to the intermolecular ET problem. This procedure is extremely efficient because it targets only the functionally active complex geometries by introducing a "reactivity filter" into the computations themselves, rather than as a subsequent step. This efficiency allows us to employ more computationally expensive and accurate methods to describe the relevant intermolecular interaction energies and the protein-mediated donor/acceptor coupling interactions. It is employed here to compute the changes in the bimolecular rate constant for ET between Mb and cyt b(5) upon variations in the myoglobin surface charge, pH, and ionic strength.

We present a broad study of the effect of neutralizing the two negative charges of the Mb propionates on the interaction and electron transfer (ET) between horse Mb and bovine cyt b(5), through use of Zn-substituted Mb (ZnMb, 1) to study the photoinitiated reaction, ((3)ZnP)Mb + Fe(3+)cyt b(5) --> (ZnP)(+)Mb + Fe(2+)cyt b(5). The charge neutralization has been carried out both by replacing the Mb heme with zinc-deuteroporphyrin dimethylester (ZnMb(dme), 2), which replaces the charges by small neutral hydrophobic patches, and also by replacement with the newly prepared zinc-deuteroporphyrin diamide (ZnMb(diamide), 3), which converts the charged groups to neutral, hydrophilic ones. The effect of propionate neutralization on the conformation of the zinc-porphyrin in the Mb heme pocket has been studied by multinuclear NMR with an (15)N labeled zinc porphyrin derivative (ZnMb((15)N-diamide), 4). The rates of photoinitiated ET between the Mb's (1-3) and cyt b(5) have been measured over a range of pH values and ionic strengths. Isothermal titration calorimetry (ITC) and NMR methods have been used to independently investigate the effect of charge neutralization on Mb/b(5) binding. The neutralization of the two heme propionates of ZnMb by formation of the heme diester or, for the first time, the diamide increases the second-order rate constant of the ET reaction between ZnMb and cyt b(5) by as much as several 100-fold, depending on pH and ionic strength, while causing negligible changes in binding affinity. Brownian dynamic (BD) simulations and ET pathway calculations provide insight into the protein docking and ET process. The results support a new "dynamic docking" paradigm for protein-protein reactions in which numerous weakly bound conformations of the docked complex contribute to the binding of cyt b(5) to Mb and Hb, but only a very small subset of these are ET active, and this subset does not include the conformations most favorable for binding; the Mb surface is a large "target" with a small "bullseye" for the cyt b(5) "arrow". This paradigm differs sharply from the more familiar, "simple" docking within a single, or narrow range of conformations, where binding strength and ET reactivity increase in parallel. Likewise, it is distinct from, although complementary to, the well-known picture of conformational control of ET through "gating", or a related picture of "conformational coupling". The new model describes situations in which tight binding does not correlate with efficient ET reactivity, and explains how it is possible to modulate reactivity without changing affinity. Such "decoupling" of reactivity from binding clearly is of physiological relevance for the reduction of met-Mb in muscle and of met-Hb in a red cell, where tight binding of cyt b(5) to the high concentration of ferrous-Mb/Hb would prevent the cytochrome from finding and reducing the oxidized proteins; it likely is of physiological relevance in other situations, as well.

The interaction of trypsin-digested bovine cytochrome b(5) (cyt b(5)) with horse heart myoglobin (Mb) and the interprotein electron transfer (ET) between these redox partners have been studied to gain better understanding of ET processes between weakly bound protein partners. The bimolecular rate constant ( k(2)) for photo-induced ET between zinc-substituted Mb (ZnMb) and cyt b(5) decreases with increasing ionic strength, consistent with the predominantly electrostatic character of this complex. The formation of a protein-protein complex has been confirmed and the binding affinities of metMb and ZnMb for cyt b(5) have been measured by two techniques: (1)H NMR titrations at pH 6.0 give binding constants of K(a) approximately (1.0+/-0.1)x10(3) M(-1) for metMb and K(a) approximately (0.75+/-0.1)x10(3) M(-1) for ZnMb; isothermal calorimetry gives K(a) approximately (0.35+/-0.1)x10(3) M(-1) for ZnMb. Brownian dynamic (BD) simulations show that cyt b(5) binds over a broad surface of Mb that includes its heme edge. The experimental results are described in terms of a dynamic docking model which proposes that Mb binds cyt b(5) in a large ensemble of protein binding conformations, not one or a few dominant ones, but that only a small subset are ET reactive. Aided by the BD simulations, this model explains why k(2) decreases with increasing pH: increasing pH not only weakens the binding affinity but also reduces the number of binding conformations with high ET reactivity.