Polyphosphate Plays a Significant Role in the Maturation of Spores in Myxococcus xanthus.

Myxococcus xanthus synthesizes polyphosphates (polyPs) with polyphosphate kinase 1 (Ppk1) and degrades short- and long-chain polyPs with the exopolyphosphatases, Ppx1 and Ppx2, respectively. M. xanthus polyP:AMP phosphotransferase (Pap) generates ADP from AMP and polyPs. Pap expression is induced by an elevation in intracellular polyP concentration. M. xanthus synthesized polyPs during the stationary phase; the ppk1 mutant died earlier than the wild-type strain after the stationary phase. In addition, M. xanthus cells cultured in phosphate-starved medium, H2O2-supplemented medium, or amino acid-deficient medium increased the intracellular polyP levels by six- to ninefold after 6 h of incubation. However, the growth of ppk1 and ppx2 mutants in phosphate-starved medium and H2O2-supplemented medium was not significantly different from that of wild-type strain, nor was there a significant difference in fruiting body formation and sporulation in starvation condition. During development, no difference was observed in the adenylate energy charge (AEC) values in the wild-type, ppk1 mutant, and pap mutant strains until the second day of development. However, after day 3, the ppk1 and pap mutants had a lower ADP ratio and a higher AMP ratio compared to wild-type strain, and as a result, the AEC values of these mutants were lower than those of the wild-type strain. Spores of ppk1 and pap mutants in the nutrient medium germinated later than those of the wild-type strain. These results suggested that polyPs produced during development may play an important role in cellular energy homeostasis of the spores by being used to convert AMP to ADP via Pap.

Synthesis and degradation of polyphosphate in Myxococcus xanthus.

Polyphosphate kinase 1 (Ppk1) generates polyphosphates (polyPs) by catalyzing phosphate transfer from ATP. In the presence of ATP, Myxococcus xanthus Ppk1 showed the highest activity with polyP60-70 but also showed high activity with orthophosphate and pyrophosphate. Ppk1 synthesizes long-chain polyPs with >1 000 phosphate residues from orthophosphate or pyrophosphate present in high concentrations, suggesting that in M. xanthus, Ppk1 uses intracellular ortho/pyrophosphate as an initial primer for polyP production. During M. xanthus starvation-induced development, the specific activity of Ppk1 peaked at 12 h (300-800 nmol/min/mg) and then gradually decreased. The polyP concentration was highest during mound formation (45 nmol phosphate/mg protein); then, the level of long-chain polyPs decreased and that of short-chain polyPs increased during fruiting body and spore formation. Myxococcus xanthus expresses two exopolyphosphatases, Ppx1 and Ppx2, which mainly degrade short- and long-chain polyPs, respectively, both of which were highest in vegetative cells and were detected during starvation, which may account for the degradation of polyPs. Thus, polyPs synthesized by Ppk1 early in starvation-induced development could be degraded by exopolyphosphatases and may also be used as substrates by polyP:AMP phosphotransferases and polyphosphate/ATP-NAD kinases to generate ADP and NADP+, respectively.

Characterization of glutamate-cysteine ligase and glutathione synthetase from the δ-proteobacterium Myxococcus xanthus.

Glutathione (GSH) is synthesized in two ATP-dependent reactions by glutamate-cysteine ligase (Gcl) and glutathione synthetase (Gs). Myxococcus xanthus, a gram-negative bacterium belonging to δ-proteobacteria, possesses mxGcl and mxGs, which have high sequence identity with the enzymes from plants and bacteria, respectively. MxGcl2 was activated by Mn2+ , but not by Mg2+ , and stabilized in the presence of 5 mM Mn2+ or Mg2+ . Sequence comparison of mxGcl2 and Brassica juncea Gcl indicated that they have the same active site residues, except for Tyr330, which interacts with Cys and which in mxGcl2 is represented by Leu267. The substitution of Leu267 with Tyr resulted in the loss of mxGcl2 activity, but that with Met (found in cyanobacterial Gcls) increased the mxGcl2 affinity for Cys. GSH and its oxidized form GSSG equally inhibited the activity of mxGcl2; the inhibition was augmented by ATP at concentrations >3 mM. Buthionine sulfoximine inactivated mxGcl2 with Ki  = 2.1 µM, which was lower than those for Gcls from other organisms. The mxGcl2 activity was also suppressed by pyrophosphate and polyphosphates. MxGs was a dimer, and its activity was induced by Mg2+ but strongly inhibited by Mn2+ even in the presence of 10 mM Mg2+ . MxGs was inhibited by GSSG at Ki  = 3.6 mM. Approximately 1 mM GSH was generated with 3 units of mxGcl2 and 6 units of mxGs from 5 mM Glu, Cys, and Gly, and 10 mM ATP. Our results suggest that GSH production in M. xanthus mostly depends on mxGcl2 activity.

Enzymatic properties of Myxococcus xanthus exopolyphosphatases mxPpx1 and mxPpx2.

Myxococcus xanthus possesses two exopolyphosphatases, mxPpx1 and mxPpx2, which belong to the family of Ppx/GppA phosphatases; however, their catalytic properties have not been described. mxPpx1 and mxPpx2 contain 311 and 505 amino acid residues, respectively; mxPpx2 has an additional C-terminal region, which corresponds to the metal-dependent HDc phosphohydrolase domain. mxPpx1 mainly hydrolyzed short-chain polyPs (polyP3 and polyP4), whereas mxPpx2 preferred long-chain polyP60-70 and polyP700-1000. mxPpx2 was activated by 25-50 mM KCl, but mxPpx1 did not significantly depend on K+. In addition, mxPpx1 and mxPpx2 showed weak hydrolysis of ATP and GTP in the absence of K+, and mxPpx2 could also hydrolyze guanosine pentaphosphate (pppGpp) in the presence of K+. The exopolyphosphatase activity of mxPpx1 toward polyP3 was inhibited by polyP700-1000 and that of mxPpx2 toward polyP60-70 and polyP700-1000, by pyrophosphate. To clarify the function of the mxPpx2 C-terminal domain, it was fused to mxPpx1 (mxPpx1-2C) and deleted from mxPpx2 (mxPpx2∆C). Compared to wild-type mxPpx2, mxPpx2∆C had significantly reduced exopolyphosphatase activity toward long-chain polyPs (by 90%), whereas that toward polyP3 and polyP4 was much less affected; furthermore, the phosphohydrolase activity toward pppGpp, ATP, and GTP was also decreased (by 30-75%). In contrast, mxPpx1-2C had increased hydrolytic activity compared to mxPpx1. Furthermore, mxPpx2∆C lost the requirement for K+ characteristic for the wild-type enzyme, whereas mxPpx1-2C acquired it. These results suggest that the C-terminal domain of mxPpx2 is necessary for its maximum hydrolytic activity, especially toward long-chain polyPs, and defines mxPpx2 dependency on K+ for activation.

No adverse events were observed in clozapine-treated patients on extended hematologic monitoring intervals during the coronavirus pandemic in four psychiatric centers in Japan.

AIM: As an emergency measure during the coronavirus disease pandemic, the monitoring interval for clozapine use was temporarily extended beyond the regulatory requirement in Japan, which is the safest monitoring interval worldwide. In this study, we aimed to explore the effect of this measure on patients undergoing clozapine treatment. METHODS: This retrospective chart review study included patients with treatment-resistant schizophrenia (TRS) who were undergoing clozapine treatment at four psychiatric institutions in Japan. Demographic characteristics and clinical information of these patients were collected on April 27, 2020, when Japanese psychiatrists were virtually allowed to prescribe clozapine beyond the regulatory requirement. Furthermore, information of adverse events related to the emergency measure was collected and analyzed. RESULTS: Of the 41 patients with TRS included in this study, 19 patients underwent extended hematological monitoring during clozapine treatment. No psychiatric or hematological adverse events were observed in the patients during the extended monitoring interval. CONCLUSION: This study suggested that there were few adverse events of clozapine-treated patients related to emergency measures in Japan. However, hematological monitoring intervals during clozapine treatment have been emergently extended worldwide; hence, it is necessary to verify the results of these measures.

Catalytic activity profile of polyP:AMP phosphotransferase from Myxococcus xanthus.

Myxococcus xanthus generates polyphosphates (polyPs) during starvation and forms fruiting bodies through the activity of polyphosphate kinase (Ppk). M. xanthus polyP:AMP phosphotransferase (Pap), a class II Ppk2, catalyzes the transfer of the terminal phosphate from polyP to AMP to yield ADP, but its enzymatic properties have not been investigated in detail. In this study, we found that Pap was activated by Mn2+ or Mg2+ and required higher concentrations of these ions in reactions with longer polyPs to demonstrate maximum activity. The Km of Pap for polyP700-1000 was significantly lower than that for shorter polyPs, but the highest catalytic constant (kcat) was observed for polyP60-70. When Pap was incubated with polyP60-70 and AMP for 3 h, it first generated ADP and then gradually produced ATP, suggesting that M. xanthus Pap also has polyP:ADP phosphotransferase activity similar to that of class III Ppk2 enzymes. During starvation, the specific activity of Pap in M. xanthus was increased by 2.3-2.4-fold at days 1 and 2 of incubation. In addition, recombinant Pap in combination with M. xanthus recombinant enzymes Ppk1 or adenylate kinase (AdkA) could generate ATP from AMP and polyP60-70. These results suggest a functional role of Pap during M. xanthus starvation, when it might act in cooperation with Ppk1 and/or AdkA to produce ATP from AMP, ADP, and polyP.

Enzymatic characteristics of Nudix hydrolase 2 (Nud2), an 8-oxo-dGTP hydrolase from Myxococcus xanthus.

Myxococcus xanthus Nudix hydrolase 2 (Nud2) hydrolyzed oxidized deoxynucleotides, such as 8-oxo-dGTP, 8-oxo-dGDP, 8-OH-dTP, and 2-OH-dATP, and showed the highest specific activity toward 8-oxo-dGTP. Mn2+ was the most effective co-factor for stimulating oxidized deoxynucleotide hydrolase activity. The Km of Nud2 with 8-oxo-dGTP for Mn2+ was 19-fold lower than that for Mg2+, and was 2-fold lower than that with dGTP for Mn2+. The specificity constant (kcat/Km) for 8-oxo-dGTP was 6-fold higher than that for dGTP. Nud2 contains a similar Nudix motif (84AX590GX7REX2EEXGX). Replacement of Ala84 and/or Gly90 in the Nudix motif of Nud2 by Gly or Glu had negligible effects on 8-oxo-dGTP hydrolase activity, suggesting that a strict Nudix motif sequence is not essential for complete hydrolase activity of Nud2.

Enzymatic Characteristics of a Polyphosphate/ATP-NAD Kinase, PanK, from Myxococcus xanthus.

NAD kinase is a crucial enzyme for production of NADP+. Myxococcus xanthus is a gram-negative soil bacterium that forms fruiting bodies and spores under starvation, and it accumulates polyphosphate (poly(P)) during early development. We found that M. xanthus NAD kinase (PanK) utilized both ATP and poly(P) as phosphoryl donors; therefore, PanK was designated as a poly(P)/ATP-NAD kinase. Unlike other poly(P)/ATP-NAD kinases, PanK hardly exhibited NADH kinase activity. The NAD kinase activity of PanK was inhibited by NADPH, but not NADH. Replacement of Thr-90 in the GGDGT motif of PanK with Asn decreased both ATP- and poly(P)-dependent NAD kinase activities; however, poly(P)-dependent NAD kinase activity was further decreased by approximately 6- to 10-fold compared with ATP-dependent NAD kinase activity, suggesting that Thr-90 in the GGDGT motif of PanK may be important for poly(P) utilization. PanK preferred ATP and short-chain poly(P) as phosphoryl donors. The Km of PanK for ATP, poly(P)4, and poly(P)10-15 was 0.66 mM, 0.08 mM, and 0.71 mM, respectively, and the catalytic efficiency (kcat/Km) for poly(P)4 was 2.4-fold higher than that for ATP, suggesting that M. xanthus under starvation conditions may be able to efficiently generate NADP+ using PanK, ATP, and poly(P).

Enzymatic characteristics of two adenylate kinases, AdkA and AdkB, from Myxococcus xanthus.

Adenylate kinase (Adk) plays a critical role in energy metabolism and adaptation of bacteria to environmental stresses. We have previously shown that Myxococcus xanthus expresses polyphosphate kinase 1 (Ppk1) that also has Adk activity in the absence of polyphosphates. In this study, we investigated the Adk activity of the other two M. xanthus enzymes, AdkA and AdkB. The activity of AdkA was increased by dithiothreitol (DTT), which also enhanced enzyme stability. Site-directed mutagenesis of three cysteine residues (C130, C150, and C153) present in the LID domain of AdkA revealed that the Adk activity and stability of C150S and C153S mutants were not affected by DTT addition, suggesting formation of a disulfide bond between C150 and C153 in AdkA. The Km of AdkA for AMP was 8 and 17 times lower than that for ADP and ATP, respectively. AdkB is a polyphosphate kinase 2 (Ppk2) homolog lacking the Ppk2 middle region and, consequently, Ppk activity. According to our analysis, AdkB also had Adk activity and its affinity for substrates was higher than that of AdkA. Thus, M. xanthus expresses three enzymes, AdkA, AdkB, and Ppk1, with Adk activity, which may function to support energy metabolism of the bacteria in different environmental conditions.

Identification of Major Enzymes Involved in the Synthesis of Diadenosine Tetraphosphate and/or Adenosine Tetraphosphate in Myxococcus xanthus.

Myxococcus xanthus generates diadenosine tetraphosphates (Ap4A) and diadenosine pentaphosphates (Ap5A) under various stress conditions. M. xanthus lysyl-tRNA synthetase (LysS) efficiently synthesizes Ap4A from ATP, Ap5A from ATP and adenosine tetraphosphate (Ap4), and Ap4 from ATP and triphosphate. To identify other M. xanthus enzymes that can catalyze Ap4A and Ap4 synthesis, 15 M. xanthus aminoacyl-tRNA synthetases (aaRSs), four acyl-CoA synthetases (Acys), three acetyl-CoA synthetases (Aces), phosphoglycerate kinase (Pgk), and adenylate kinase (Adk) were expressed in Escherichia coli and examined for Ap4A or Ap4 synthetase activity using ATP or ATP and triphosphate as substrates. Among the tested enzymes, LysS had the highest Ap4A synthetase activity. AlaRS, SerRS, and LeuRS1 showed high ADP synthetase activity with ATP as a substrate in the presence of pyrophosphatase, and also demonstrated the ability to produce Ap4 from ATP and triphosphate in the absence of pyrophosphatase. Ap4 formation by AlaRS, SerRS, and LeuRS1 was approximately 4- to 13-fold higher compared with that of Ap4A, suggesting that these enzymes prefer triphosphate over ATP as a substrate in the second reaction. Some of the recombinant M. xanthus Acys and Aces also synthesized Ap4 from ATP and triphosphate. However, Pgk was capable of catalyzing the production of Ap4 from ATP and 3-phosphoglycerate in the presence of Mg2+ and did not require triphosphate, suggesting that this enzyme is mainly responsible for Ap4 synthesis in M. xanthus.

Catalytic Activity Profile of Polyphosphate Kinase 1 from Myxococcus xanthus.

Polyphosphate kinase 1 (Ppk1) catalyzes reverse transfer of the terminal phosphate from ATP to form polyphosphate (polyP) and from polyP to form ATP, and is responsible for the synthesis of most of cellular polyPs. When Ppk1 from Myxococcus xanthus was incubated with 0.2 mM polyP60-70 and 1 mM ATP or ADP, the rate of ATP synthesis was approximately 1.5-fold higher than that of polyP synthesis. If in the same reaction the proportion of ADP in the ATP/ADP mixture exceeded one-third, the equilibrium shifted to ATP synthesis, suggesting that M. xanthus Ppk1 preferentially catalyzed ATP formation. At the same time, GTP and GDP were not recognized as substrates by Ppk1. In the absence of polyP, Ppk1 generated ATP and AMP from ADP, and ADP from ATP and AMP, suggesting that the enzyme catalyzed the transfer of a phosphate group between ADP molecules yielding ATP and AMP, thus exhibiting adenylate kinase activity.

High concentrations of intracellular Ap4A and/or Ap5A in developing Myxococcus xanthus cells inhibit sporulation.

Diadenosine polyphosphates (ApnA) are thought to act as signalling molecules regulating stress responses and biofilm formation in prokaryotes. However, ApnA function in Myxococcus xanthus remains unknown. Here, we investigated the role of ApnA in M. xanthus, using the wild-type and ApnA hydrolase (apaH) mutant strains exposed to various stress conditions. In both wild-type and apaH mutant cells cultured on starvation medium (CF agar), the levels of intracellular diadenosine tetraphosphate (Ap4A) and pentaphosphate (Ap5A) increased several fold during the first 16 h of development and decreased gradually thereafter. The levels of Ap4A and Ap5A in the apaH mutant were about 5- and 11-fold higher than those in the wild-type strain at 16 h, respectively. ApnA hydrolase activity of the wild-type strain increased 1.5-fold during the first 8 h of development, and it then gradually decreased. The apaH mutant formed spores 1-2 days after the wild-type strain did, and the yield of viable spores was 5.5 % of that in the wild-type strain 5 days after inoculation onto CF agar. These results suggest the possibility that high intracellular levels of Ap4A and/or Ap5A may inhibit M. xanthus sporulation at the early stage of development and that the bacteria reduce intracellular Ap4A and Ap5A accumulation through ApnA hydrolase activity.

Lysyl-tRNA synthetase from Myxococcus xanthus catalyzes the formation of diadenosine penta- and hexaphosphates from adenosine tetraphosphate.

Myxococcus xanthus lysyl-tRNA synthetase (LysS) produces diadenosine tetraphosphate (Ap4A) from ATP in the presence of Mn(2+); in the present study, it also generated Ap4 from ATP and triphosphate. When ATP and Ap4 were incubated with LysS and pyrophosphatase, first Ap4A, Ap5A, and ADP, and then Ap5, Ap6A, and Ap3A were generated. The results suggest that in the first step, LysS can form lysyl-AMP and lysyl-ADP intermediates from Ap4 and release triphosphate and diphosphate, respectively, whereas in the second step, it can produce Ap5 from lysyl-ADP with triphosphate, and Ap6A from lysyl-ADP with Ap4. In addition, in the presence of Ap4 and pyrophosphatase, but absence of ATP, LysS also generates diadenosine oligophosphates (ApnAs: n = 3-6). These results indicate that LysS has the ability to catalyze the formation of various ApnAs from Ap4 in the presence of pyrophosphatase. Furthermore, the formation of Ap4A by LysS was inhibited by tRNA(Lys) in the presence of 1 mM ATP. To the best of our knowledge, this is the first report of Ap5A and Ap6A synthesis by lysyl-tRNA synthetase.

Characterization of a eukaryotic-like protein kinase, DspB, with an atypical catalytic loop motif from Myxococcus xanthus.

Serine (Ser)/threonine (Thr) or tyrosine (Tyr) protein kinases in eukaryotes contain RDxKxxN or RDx(A/R)A(A/R)N sequences, respectively, in the catalytic loop. Myxococcus xanthus DspB is a dual-specificity kinase that contains an atypical sequence, RDVAQKN, in the catalytic loop. The DspB mutant (A165K), which contains the canonical RDxKxxN motif, had an approximate 1.3-fold increase in kinase activity toward myelin basic protein (MBP). Arginine-aspartate (RD) kinases carry a conserved Arg immediately preceding the catalytic Asp that is required for autophosphorylation of the activation loop. DspB belongs to the RD kinase family and contains one Ser residue (Ser-190) and one Thr residue (Thr-194) in the activation loop. Mutation of Ser-190 or Thr-194 to Ala did not significantly affect the kinase activity toward MBP. We previously reported that four M. xanthus eukaryotic-like kinases (EPKs) are autophosphorylated on Tyr residues. These EPKs contain six Tyr residues at homologous positions, and five of those Tyr residues, Y25, Y102, Y145, Y173, and Y205, are conserved in DspB. DspB is mainly autophosphorylated on Y145, and a Y145F mutant has reduced kinase activity, suggesting that autophosphorylation of the Tyr residue of DspB may be required for high-level kinase activity.

Enzymatic characterization of a class II lysyl-tRNA synthetase, LysS, from Myxococcus xanthus.

Lysyl-tRNA synthetases efficiently produce diadenosine tetraphosphate (Ap4A) from lysyl-AMP with ATP in the absence of tRNA. We characterized recombinant class II lysyl-tRNA synthetase (LysS) from Myxococcus xanthus and found that it is monomeric and requires Mn(2+) for the synthesis of Ap4A. Surprisingly, Zn(2+) inhibited enzyme activity in the presence of Mn(2+). When incubated with ATP, Mn(2+), lysine, and inorganic pyrophosphatase, LysS first produced Ap4A and ADP, then converted Ap4A to diadenosine triphosphate (Ap3A), and finally converted Ap3A to ADP, the end product of the reaction. Recombinant LysS retained Ap4A synthase activity without lysine addition. Additionally, when incubated with Ap4A (minus pyrophosphatase), LysS converted Ap4A mainly ATP and AMP, or ADP in the presence or absence of lysine, respectively. These results demonstrate that M. xanthus LysS has different enzymatic properties from class II lysyl-tRNA synthetases previously reported.

Functional analysis of conserved motifs in a bacterial tyrosine kinase, BtkB, from Myxococcus xanthus.

Myxococcus xanthus has two bacterial protein-tyrosine (BY) kinases, BtkA and BtkB. Autophosphorylation in C-terminal tyrosine-rich clusters and poly(Glu, Tyr) kinase activities of cytoplasmic catalytic domains of BtkA and BtkB were activated by the intracellular juxtamembrane regions of the second transmembrane helices. Protein kinase activity against poly(Glu, Tyr) of cytoplasmic fragment of BtkB (CF-BtkB) containing an activator region was not inhibited by serine/threonine protein kinase inhibitors. However, addition of tyrosine protein kinase inhibitors, genistein and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2), at a concentration of 0.2 mM, inhibited the CF-BtkB kinase activity by 20 and 64%, respectively. A CF-BtkB mutant constructed by replacing all C-terminal tyrosine residues with phenylalanines, did not undergo autophosphorylation. Further, this mutation did not significantly affect poly(Glu, Tyr) kinase activity, suggesting that M. xanthus BtkB kinase activity is not dependent on autophosphorylation in the C-terminal tyrosine cluster. A conserved motif (ExxRxxR) of BY kinases is involved in the self-association of catalytic domains of BY kinases, necessary to accomplish trans-phosphorylation. An ExxRxxR motif mutant of CF-BtkB led to loss of autophosphorylation and poly(Glu, Tyr) kinase activities. These observations provide insights into the regulation mechanism of M. xanthus BY kinase activity.

Characterizing activities of eukaryotic-like protein kinases with atypical catalytic loop motifs from Myxococcus xanthus.

Myxococcus xanthus has eukaryotic-like protein kinases (EPKs) with different atypical catalytic loop motifs. Seven out of 14 recombinant M. xanthus EPKs containing atypical motifs in the catalytic loop showed protein kinase activity against myelin basic protein and four autophosphorylated EPKs were detected using anti-phosphotyrosine antibody by western blotting.

Enzymatic characteristics of an ApaH-like phosphatase, PrpA, and a diadenosine tetraphosphate hydrolase, ApaH, from Myxococcus xanthus.

We characterized the activities of the Myxococcus xanthus ApaH-like phosphatases PrpA and ApaH, which share homologies with both phosphoprotein phosphatases and diadenosine tetraphosphate (Ap4A) hydrolases. PrpA exhibited a phosphatase activity towards p-nitrophenyl phosphate (pNPP), tyrosine phosphopeptide and tyrosine-phosphorylated protein, and a weak hydrolase activity towards ApnA and ATP. In the presence of Mn(2+), PrpA hydrolyzed Ap4A into AMP and ATP, whereas in the presence of Co(2+) PrpA hydrolyzed Ap4A into two molecules of ADP. ApaH exhibited high phosphatase activity towards pNPP, and hydrolase activity towards ApnA and ATP. Mn(2+) was required for ApaH-mediated pNPP dephosphorylation and ATP hydrolysis, whereas Co(2+) was required for ApnA hydrolysis. Thus, PrpA and ApaH may function mainly as a tyrosine protein phosphatase and an ApnA hydrolase, respectively.