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The Gram-negative bacterium Myxococcus xanthus glides on solid surfaces. Dynamic bacterial focal adhesion complexes (bFACs) convert proton motive force from the inner membrane into mechanical propulsion on the cell surface. It is unclear how the mechanical force transmits across the rigid peptidoglycan (PG) cell wall. Here, we show that AgmT , a highly abundant lytic PG transglycosylase homologous to Escherichia coli MltG, couples bFACs to PG. Coprecipitation assay and single-particle microscopy reveal that the gliding motors fail to connect to PG and thus are unable to assemble into bFACs in the absence of an active AgmT. Heterologous expression of E. coli MltG restores the connection between PG and bFACs and thus rescues gliding motility in the M. xanthus cells that lack AgmT. Our results indicate that bFACs anchor to AgmT-modified PG to transmit mechanical force across the PG cell wall., Competing Interests: CR, OF, BN No competing interests declared, (© 2024, Ramirez Carbo, Faromiki et al.)

Encapsulins are protein nanocompartments that regulate cellular metabolism in several bacteria and archaea. Myxococcus xanthus encapsulins protect the bacterial cells against oxidative stress by sequestering cytosolic iron. These encapsulins are formed by the shell protein EncA and three cargo proteins: EncB, EncC, and EncD. EncB and EncC form rotationally symmetric decamers with ferroxidase centers (FOCs) that oxidize Fe +2 to Fe +3 for iron storage in mineral form. However, the structure and function of the third cargo protein, EncD, have yet to be determined. Here, we report the x-ray crystal structure of EncD in complex with flavin mononucleotide. EncD forms an α-helical hairpin arranged as an antiparallel dimer, but unlike other flavin-binding proteins, it has no β-sheet, showing that EncD and its homologs represent a unique class of bacterial flavin-binding proteins. The cryo-EM structure of EncA-EncD encapsulins confirms that EncD binds to the interior of the EncA shell via its C-terminal targeting peptide. With only 100 amino acids, the EncD α-helical dimer forms the smallest flavin-binding domain observed to date. Unlike EncB and EncC, EncD lacks a FOC, and our biochemical results show that EncD instead is a NAD(P)H-dependent ferric reductase, indicating that the M. xanthus encapsulins act as an integrated system for iron homeostasis. Overall, this work contributes to our understanding of bacterial metabolism and could lead to the development of technologies for iron biomineralization and the production of iron-containing materials for the treatment of various diseases associated with oxidative stress., Competing Interests: Competing interests statement:The authors declare no competing interest.

Cell polarity oscillations in Myxococcus xanthus motility are driven by a prokaryotic small Ras-like GTPase, mutual gliding protein A (MglA), which switches from one cell pole to the other in response to extracellular signals. MglA dynamics is regulated by MglB, which functions both as a GTPase activating protein (GAP) and a guanine nucleotide exchange factor (GEF) for MglA. With an aim to dissect the asymmetric role of the two MglB protomers in the dual GAP and GEF activities, we generated a functional MglAB complex by coexpressing MglB with a linked construct of MglA and MglB. This strategy enabled us to generate mutations of individual MglB protomers (MglB 1 or MglB 2 linked to MglA) and delineate their role in GEF and GAP activities. We establish that the C-terminal helix of MglB 1 , but not MglB 2 , stimulates nucleotide exchange through a site away from the nucleotide-binding pocket, confirming an allosteric mechanism. Interaction between the N-terminal β-strand of MglB 1 and β 0 of MglA is essential for the optimal GEF activity of MglB. Specific residues of MglB 2, which interact with Switch-I of MglA, partially contribute to its GAP activity. Thus, the role of the MglB 2 protomer in the GAP activity of MglB is limited to restricting the conformation of MglA active site loops. The direct demonstration of the allosteric mechanism of GEF action provides us new insights into the regulation of small Ras-like GTPases, a feature potentially present in many uncharacterized GEFs., Competing Interests: Conflict of interest The authors declare no conflict of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Thioredoxin (Trx) is a disulfide-containing redox protein that functions as a disulfide oxidoreductase. Myxococcus xanthus contains five Trxs (Trx1-Trx5) and one Trx reductase (TrxR). Trxs typically have a CGPC active-site motif; however, M. xanthus Trxs have slightly different active-site sequences, with the exception of Trx4. The five Trxs of M. xanthus exhibited reduced activities against insulin, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), cystine, glutathione disulfide (GSSG), S-nitrosoglutathione (GSNO), and H2O2 in the presence of TrxR. Myxococcus xanthus adenylate kinase and serine/threonine phosphatase activities, which were increased by the addition of dithiothreitol, were activated by the addition of Trxs and TrxR. Among these, Trx1, which has a CAPC sequence in its active site, exhibited the highest reducing activity with the exception of GSNO. Myxococcus xanthus TrxR showed weak reducing activity towards DTNB, GSSG, GSNO, and H2O2, suggesting that it has broad substrate specificity, unlike previously reported low-molecular-weight TrxRs. TrxR reduced oxidized Trx1 as the best substrate, with a kcat/Km value of 0.253 min-1 µM-1, which was 10-28-fold higher than that of the other Trxs. These results suggest that all Trxs possess reducing activity and that Trx1 may be the most functional in M. xanthus because TrxR most efficiently reduces oxidized Trx1., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

Motile cells have developed a large array of molecular machineries to actively change their direction of movement in response to spatial cues from their environment. In this process, small GTPases act as molecular switches and work in tandem with regulators and sensors of their guanine nucleotide status (GAP, GEF, GDI and effectors) to dynamically polarize the cell and regulate its motility. In this review, we focus on Myxococcus xanthus as a model organism to elucidate the function of an atypical small Ras GTPase system in the control of directed cell motility. M. xanthus cells direct their motility by reversing their direction of movement through a mechanism involving the redirection of the motility apparatus to the opposite cell pole. The reversal frequency of moving M. xanthus cells is controlled by modular and interconnected protein networks linking the chemosensory-like frizzy (Frz) pathway - that transmits environmental signals - to the downstream Ras-like Mgl polarity control system - that comprises the Ras-like MglA GTPase protein and its regulators. Here, we discuss how variations in the GTPase interactome landscape underlie single-cell decisions and consequently, multicellular patterns., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

ParB-like CTPases mediate the segregation of bacterial chromosomes and low-copy number plasmids. They act as DNA-sliding clamps that are loaded at parS motifs in the centromere of target DNA molecules and spread laterally to form large nucleoprotein complexes serving as docking points for the DNA segregation machinery. Here, we solve crystal structures of ParB in the pre- and post-hydrolysis state and illuminate the catalytic mechanism of nucleotide hydrolysis. Moreover, we identify conformational changes that underlie the CTP- and parS-dependent closure of ParB clamps. The study of CTPase-deficient ParB variants reveals that CTP hydrolysis serves to limit the sliding time of ParB clamps and thus drives the establishment of a well-defined ParB diffusion gradient across the centromere whose dynamics are critical for DNA segregation. These findings clarify the role of the ParB CTPase cycle in partition complex assembly and function and thus advance our understanding of this prototypic CTP-dependent molecular switch., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

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 (polyP 3 and polyP 4 ), whereas mxPpx2 preferred long-chain polyP 60 - 70 and polyP 700 - 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 polyP 3 was inhibited by polyP 700 - 1000 and that of mxPpx2 toward polyP 60 - 70 and polyP 700 - 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 polyP 3 and polyP 4 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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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 Mn 2+ or Mg 2+ and required higher concentrations of these ions in reactions with longer polyPs to demonstrate maximum activity. The K m of Pap for polyP 700-1000 was significantly lower than that for shorter polyPs, but the highest catalytic constant (k cat ) was observed for polyP 60-70 . When Pap was incubated with polyP 60-70 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 polyP 60-70 . 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., (Copyright © 2020 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The enzyme 3-methylglutaconyl coenzyme A (CoA) decarboxylase (called AibA/AibB) catalyzes the decarboxylation of 3-methylglutaconyl CoA to generate 3,3-dimethylacrylyl-CoA, representing an important step in the biosynthesis of isovaleryl-coenzyme A in Myxococcus xanthus when the regular pathway is blocked. A novel mechanism involving a pericyclic transition state has previously been proposed for this enzyme, making AibA/AibB unique among decarboxylases. Herein, density functional calculations are used to examine the energetic feasibility of this mechanism. It is shown that the intramolecular pericyclic reaction is associated with a very high energy barrier that is similar to the barrier of the same reaction in the absence of the enzyme. Instead, the calculations show that a direct decarboxylation mechanism has feasible energy barriers that are in line with the experimental observations., (© 2020 The Authors. Published by Wiley-VCH GmbH.)

Objective: To produce high concentrations of 13-hydroxy-14,15-epoxy-eicosatrienoic acid (14,15-hepoxilin B 3 , 14,15-HXB 3 ) and 13,14,15-trihydroxy-eicosatrienoic acid (13,14,15-trioxilin B 3 , 13,14,15-TrXB 3 ) from arachidonic acid (ARA) using microbial 15-lipoxygenase (15-LOX) without and with epoxide hydrolase (EH), respectively., Results: The products obtained from the bioconversion of ARA by recombinant Escherichia coli cells containing Archangium violaceum 15-LOX without and with Myxococcus xanthus EH were identified as 14,15-HXB 3 and 13,14,15-TrXB 3 , respectively. Under the optimal conditions of 30 g cells L -1 , 200 mM ARA, 25 °C, and initial pH 7.5, the cells converted 200 mM ARA into 192 mM 14,15-HXB 3 and 100 mM 13,14,15-TrXB 3 for 150 min, with conversion yields of 96 and 51% and productivities of 77 and 40 mM h -1 , respectively., Conclusion: These are the highest concentrations, productivities, and yields of hepoxilin and trioxilin from ARA reported thus far.

The rod-shaped cells of Myxococcus xanthus, a Gram-negative deltaproteobacterium, differentiate to environmentally resistant spores upon starvation or chemical stress. The environmental resistance depends on a spore coat polysaccharide that is synthesised by the ExoA-I proteins, some of which are part of a Wzx/Wzy-dependent pathway for polysaccharide synthesis and export; however, key components of this pathway have remained unidentified. Here, we identify and characterise two additional loci encoding proteins with homology to enzymes involved in polysaccharide synthesis and export, as well as sugar modification and show that six of the proteins encoded by these loci are essential for the formation of environmentally resistant spores. Our data support that MXAN_3260, renamed ExoM and MXAN_3026, renamed ExoJ, are the Wzx flippase and Wzy polymerase, respectively, responsible for translocation and polymerisation of the repeat unit of the spore coat polysaccharide. Moreover, we provide evidence that three glycosyltransferases (MXAN_3027/ExoK, MXAN_3262/ExoO and MXAN_3263/ExoP) and a polysaccharide deacetylase (MXAN_3259/ExoL) are important for formation of the intact spore coat, while ExoE is the polyisoprenyl-phosphate hexose-1-phosphate transferase responsible for initiating repeat unit synthesis, likely by transferring N-acetylgalactosamine-1-P to undecaprenyl-phosphate. Together, our data generate a more complete model of the Exo pathway for spore coat polysaccharide biosynthesis and export., (© 2020 The Authors. Moelecular Microbiology published by John Wiley & Sons Ltd.)

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. Mn 2+ was the most effective co-factor for stimulating oxidized deoxynucleotide hydrolase activity. The K m of Nud2 with 8-oxo-dGTP for Mn 2+ was 19-fold lower than that for Mg 2+ , and was 2-fold lower than that with dGTP for Mn 2+ . The specificity constant (k cat /K m ) for 8-oxo-dGTP was 6-fold higher than that for dGTP. Nud2 contains a similar Nudix motif ( 84 AX 5 90 GX 7 REX 2 EEXGX). 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.

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 K m 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 (k cat /K m ) 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).

3',3'-cyclic GMP-AMP (cGAMP) is the third cyclic dinucleotide (CDN) to be discovered in bacteria. No activators of cGAMP signaling have yet been identified, and the signaling pathways for cGAMP have been inferred to display a narrow distribution based upon the characterized synthases, DncV and Hypr GGDEFs. Here, we report that the ubiquitous second messenger cyclic AMP (cAMP) is an activator of the Hypr GGDEF enzyme GacB from Myxococcus xanthus. Furthermore, we show that GacB is inhibited directly by cyclic di-GMP, which provides evidence for cross-regulation between different CDN pathways. Finally, we reveal that the HD-GYP enzyme PmxA is a cGAMP-specific phosphodiesterase (GAP) that promotes resistance to osmotic stress in M. xanthus. A signature amino acid change in PmxA was found to reprogram substrate specificity and was applied to predict the presence of non-canonical HD-GYP phosphodiesterases in many bacterial species, including phyla previously not known to utilize cGAMP signaling., (© 2019 John Wiley & Sons Ltd.)

Plasmalogens are glycerophospholipids with a hallmark sn -1 vinyl ether bond. These lipids are found in animals and some bacteria and have proposed membrane organization, signaling, and antioxidant roles. We discovered the plasmanylethanolamine desaturase activity that is essential for vinyl ether bond formation in a bacterial enzyme, CarF, which is a homolog of the human enzyme TMEM189. CarF mediates light-induced carotenogenesis in Myxococcus xanthus , and plasmalogens participate in sensing photooxidative stress through singlet oxygen. TMEM189 and other animal homologs could functionally replace CarF in M. xanthus , and knockout of TMEM189 in a human cell line eliminated plasmalogens. Discovery of the human plasmanylethanolamine desaturase will spur further study of plasmalogen biogenesis, functions, and roles in disease., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Mutual gliding motility A (MglA), a small Ras-like GTPase; Mutual gliding motility B (MglB), its GTPase activating protein (GAP); and Required for Motility Response Regulator (RomR), a protein that contains a response regulator receiver domain, are major components of a GTPase-dependent biochemical oscillator that drives cell polarity reversals in the bacterium Myxococcus xanthus. We report the crystal structure of a complex of M. xanthus MglA and MglB, which reveals that the C-terminal helix (Ct-helix) from one protomer of the dimeric MglB binds to a pocket distal to the active site of MglA. MglB increases the GTPase activity of MglA by reorientation of key catalytic residues of MglA (a GAP function) combined with allosteric regulation of nucleotide exchange by the Ct-helix (a guanine nucleotide exchange factor [GEF] function). The dual GAP-GEF activities of MglB accelerate the rate of GTP hydrolysis over multiple enzymatic cycles. Consistent with its GAP and GEF activities, MglB interacts with MglA bound to either GTP or GDP. The regulation is essential for cell polarity, because deletion of the Ct-helix causes bipolar localization of MglA, MglB, and RomR, thereby causing reversal defects in M. xanthus. A bioinformatics analysis reveals the presence of Ct-helix in homologues of MglB in other bacterial phyla, suggestive of the prevalence of the allosteric mechanism among other prokaryotic small Ras-like GTPases., Competing Interests: The authors have declared that no competing interests exist.

Myxobacterial predation on bacteria has been investigated for several decades. However, their predation on fungi has received less attention. Here, we show that a novel outer membrane β-1,6-glucanase GluM from Corallococcus sp. strain EGB is essential for initial sensing and efficient decomposition of fungi during predation. GluM belongs to an unstudied family of outer membrane β-barrel proteins with potent specific activity up to 24,000 U/mg, whose homologs extensively exist in myxobacteria. GluM was able to digest fungal cell walls efficiently and restrict Magnaporthe oryzae infection of rice plants. Genetic complementation with gluM restored the fungal predation ability of Myxococcus xanthus CL1001, which was abolished by the disruption of gluM homolog oar. The inability to prey on fungi with cell walls that lack β-1,6-glucans indicates that β-1,6-glucans are targeted by GluM. Our results demonstrate that GluM confers myxobacteria with the ability to feed on fungi, and provide new insights for understanding predator-prey interactions. Considering the attack mode of GluM, we suggest that β-1,6-glucan is a promising target for the development of novel broad-spectrum antifungal agents.

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., (© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

Plant oxylipins, including hydroxy fatty acids, epoxy hydroxy fatty acids, and trihydroxy fatty acids, which are biosynthesized from C18 polyunsaturated fatty acids (PUFAs), are involved in pathogen-specific defense mechanisms against fungal infections. However, their quantitative biotransformation by plant enzymes has not been reported. A few bacteria produce C18 trihydroxy fatty acids, but the enzymes and pathways related to the biosynthesis of plant oxylipins in bacteria have not been reported. In this study, we first report the biotransformation of C18 PUFAs into plant C18 oxylipins by expressing linoleate 9 S-lipoxygenase with and without epoxide hydrolase from the proteobacterium Myxococcus xanthus in recombinant Escherichia coli. Among the nine types of plant oxylipins, 12,13-epoxy-14-hydroxy- cis, cis-9,15-octadecadienoic acid was identified as a new compound by NMR analysis, and 9,10,11-hydroxy- cis, cis-6,12-octadecadienoic acid and 12,13,14-trihydroxy- cis, cis-9,15-octadecadienoic were suggested as new compounds by LC-MS/MS analysis. This study shows that bioactive plant oxylipins can be produced by microbial enzymes.

Lipoxygenases (LOXs) catalyze the dioxygenation of PUFAs to produce regio- and stereospecific oxygenated fatty acids. The identification of regio- and stereospecific LOXs is important because their specific products are involved in different physiological activities in various organisms. Bacterial LOXs are found only in some proteobacteria and cyanobacteria, and they are not stable in vitro. Here, we used C20 and C22 PUFAs such as arachidonic acid (ARA), eicosapentaenoic acid, and docosahexaenoic acid to identify an 11 S -specific LOX from the proteobacterium Myxococcus xanthus and explore its in vitro stability and activity. The activity and stability of M. xanthus ARA 11 S -LOX as well as the production of 11 S -hydroxyeicosatetraenoic acid from ARA were significantly increased by the addition of phosphatidylcholine, Ca 2+ , and coactosin-like protein (newly identified in the yeast Rhodosporidium toluroides ) as stimulatory factors; in fact, LOX activity in the presence of all three factors increased approximately 3-fold. Our results indicate that these stimulatory factors can be used to increase the activity and stability of bacterial LOX and the production of bioactive hydroxy fatty acids, which can contribute to new academic research., (Copyright © 2018 An and Oh.)

One mechanism by which bacteria and fungi produce bioactive natural products is the use of nonribosomal peptide synthetases (NRPSs). Many NRPSs in bacteria require members of the MbtH-like protein (MLP) superfamily for their solubility or function. Although MLPs are known to interact with the adenylation domains of NRPSs, the role MLPs play in NRPS enzymology has yet to be elucidated. MLPs are nearly always encoded within the biosynthetic gene clusters (BGCs) that also code for the NRPSs that interact with the MLP. Here, we identify 50 orphan MLPs from diverse bacteria. An orphan MLP is one that is encoded by a gene that is not directly adjacent to genes predicted to be involved in nonribosomal peptide biosynthesis. We targeted the orphan MLP MXAN_3118 from Myxococcus xanthus DK1622 for characterization. The M. xanthus DK1622 genome contains 15 NRPS-encoding BGCs but only one MLP-encoding gene (MXAN_3118). We tested the hypothesis that MXAN_3118 interacts with one or more NRPS using a combination of in vivo and in vitro assays. We determined that MXAN_3118 interacts with at least seven NRPSs from distinct BGCs. We show that one of these BGCs codes for NRPS enzymology that likely produces a valine-rich natural product that inhibits the clumping of M. xanthus DK1622 in liquid culture. MXAN_3118 is the first MLP to be identified that naturally interacts with multiple NRPS systems in a single organism. The finding of an MLP that naturally interacts with multiple NRPS systems suggests it may be harnessed as a "universal" MLP for generating functional hybrid NRPSs. IMPORTANCE MbtH-like proteins (MLPs) are essential accessory proteins for the function of many nonribosomal peptide synthetases (NRPSs). We identified 50 MLPs from diverse bacteria that are coded by genes that are not located near any NRPS-encoding biosynthetic gene clusters (BGCs). We define these as orphan MLPs because their NRPS partner(s) is unknown. Investigations into the orphan MLP from Myxococcus xanthus DK1622 determined that it interacts with NRPSs from at least seven distinct BGCs. Support for these MLP-NRPS interactions came from the use of a bacterial two-hybrid assay and copurification of the MLP with various NRPSs. The flexibility of this MLP to naturally interact with multiple NRPSs led us to hypothesize that this MLP may be used as a "universal" MLP during the construction of functional hybrid NRPSs., (Copyright © 2018 American Society for Microbiology.)

Lipoxygenase (LOX) is the key enzyme involved in the synthesis of oxylipins as signaling compounds that are important for cell growth and development, inflammation, and pathogenesis in various organisms. The regiospecificity of LOX from Myxococcus xanthus, a gram-negative bacterium, was investigated. The enzyme catalyzed oxygenation at the n-9 position in C20 and C22 polyunsaturated fatty acids (PUFAs) to form 12S- and 14S-hydroxy fatty acids (HFAs), respectively, and oxygenation at the n-6 position in C18 PUFAs to form 13-HFAs. The 12S-form products of C20 and C22 PUFAs by M. xanthus LOX is the first report of bacterial LOXs. The residues involved in regiospecificity were determined to be Thr397, Ala461, and Ile664 by analyzing amino acid alignment and a homology model based on human arachidonate 15-LOX with a sequence identity of 25%. Among these variants, the regiospecificity of the T397Y variant for C20 and C22 PUFAs was changed. This may be because of the reduced size of the substrate-binding pocket by substitution of the smaller Thr to the larger Tyr residue. The T397Y variant catalyzed oxygenation at the n-6 position in C20 and C22 PUFAs to form 15- and 17-hydroperoxy fatty acids, respectively. However, the oxygenation position of T397Y for C18 PUFAs was not changed. The discovery of bacterial LOX with novel regiospecificity will facilitate the biosynthesis of regiospecific‑oxygenated signaling compounds., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The use of toxin to attack neighbours and immunity proteins to protect against toxin has been observed in bacterial conflicts, including kin discrimination. Here, we report a novel nuclease-toxin and its immunity protein function in the colony-merger incompatibility, a kind of bacterial kin discrimination, in Myxococcus xanthus DK1622. The MXAN_0049 gene was determined to be a genetic determinant for colony-merger incompatibility, and the incompatibility could be eliminated by deletion of the upstream co-transcribed MXAN_0050 gene. We demonstrated that the MXAN_0050 protein was a nuclease, and MXAN_0049 protein was able to bind to MXAN_0050 to block nuclease activity in vitro. Expression of MXAN_0050 in Escherichia coli inhibited cellular growth, and the inhibition effect could be recovered by co-expression of MXAN_0049. We found that deletion of the PAAR-encoding gene (MXAN_0044) or the type VI secretion system led to the colony-merger and co-existence with the ΔMXAN_0049 mutant, suggesting that they were associated with colony-merger incompatibility. Homologues of the nuclease-toxin and cognate immunity pair are widely distributed in bacteria. We propose a simplified model to explain the kin discrimination mechanism mediated by the nuclease-toxin and immunity protein.© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Myxococcus xanthus generates diadenosine tetraphosphates (Ap 4 A) and diadenosine pentaphosphates (Ap 5 A) under various stress conditions. M. xanthus lysyl-tRNA synthetase (LysS) efficiently synthesizes Ap 4 A from ATP, Ap 5 A from ATP and adenosine tetraphosphate (Ap 4 ), and Ap 4 from ATP and triphosphate. To identify other M. xanthus enzymes that can catalyze Ap 4 A and Ap 4 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 Ap 4 A or Ap 4 synthetase activity using ATP or ATP and triphosphate as substrates. Among the tested enzymes, LysS had the highest Ap 4 A 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 Ap 4 from ATP and triphosphate in the absence of pyrophosphatase. Ap 4 formation by AlaRS, SerRS, and LeuRS1 was approximately 4- to 13-fold higher compared with that of Ap 4 A, 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 Ap 4 from ATP and triphosphate. However, Pgk was capable of catalyzing the production of Ap 4 from ATP and 3-phosphoglycerate in the presence of Mg 2+ and did not require triphosphate, suggesting that this enzyme is mainly responsible for Ap 4 synthesis in M. 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 polyP 60-70 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.

Hepoxilins (HXs) and trioxilins (TrXs) are involved in physiological processes such as inflammation, insulin secretion and pain perception in human. They are metabolites of polyunsaturated fatty acids (PUFAs), including arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid, formed by 12-lipoxygenase (LOX) and epoxide hydrolase (EH) expressed by mammalian cells. Here, we identify ten types of HXs and TrXs, produced by the prokaryote Myxococcus xanthus, of which six types are new, namely, HXB 5 , HXD 3 , HXE 3 , TrXB 5 , TrXD 3 and TrXE 3 . We succeed in the biotransformation of PUFAs into eight types of HXs (>35% conversion) and TrXs (>10% conversion) by expressing M. xanthus 12-LOX or 11-LOX with or without EH in Escherichia coli. We determine 11-hydroxy-eicosatetraenoic acid, HXB 3 , HXB 4 , HXD 3 , TrXB 3 and TrXD 3 as potential peroxisome proliferator-activated receptor-γ partial agonists. These findings may facilitate physiological studies and drug development based on lipid mediators.

The gene encoding a novel acidic lipoxygenase from Myxococcus xanthus DK1622 (accession: WP_011551853.1) was cloned into vector pET-28a and expressed in Escherichia coli BL21(DE3). The recombinant enzyme (rMxLOX), with a molecular weight of approximately 80 kDa, was purified to homogeneity using one-step nickel-affinity chromatography and showed an activity of 5.6 × 10 4 U/mg. The optimum pH and temperature for rMxLOX activity were found to be 3.0 and 30 °C, respectively. Purified rMxLOX exhibited activity towards linoleic acid and arachidonic acid as substrates, with linoleic acid being the better substrate (K m and k cat values of 0.048 mM and 13.3/s, respectively). The synthetic dye aniline blue was decolorized 69.7 ± 3.5%, following incubation with rMxLOX for 35 min. These results reveal the potential for the use of rMxLOX in the pulp, textile, and wastewater treatment industries., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Prolyl oligopeptidase (POP, EC 3.4.21.26) is a cytosolic serine protease that hydrolyses proline containing small peptides. The members of prolyl oligopeptidase family play important roles in many physiological processes such as neurodegenerative diseases, maturation and degradation of peptide hormones. Thus the enzyme has been purified and characterized from various sources to elucidate the potential use as therapeutics. In this study recombinant Myxococcus xanthus prolyl oligopeptidase expressed in E. coli was purified 60.3 fold, using metal-chelate affinity and gel permeation chromatography. The recombinant enzyme had a monomeric molecular weight of 70 kDa. Isoelectric point of the enzyme was found to be approximately 6.3 by two-dimensional polyacrylamide gel electrophoresis. The optimum pH and temperature was estimated as 7.5 and 37 °C, respectively. The purified enzyme was stable in a pH range of 6.0-8.5 and thermally stable up to 37 °C. The K m and V max values were 0.2 mM and 3.42 μmol/min/mg. The proteolytic activity was inhibited by active-site inhibitors of serine protease, Z-Pro-Prolinal, PMSF, and metal ions, Cd 2+ , and Hg 2+ . Furthermore, the hydrolysis efficiency of the recombinant prolyl oligopeptidase was investigated with wheat gluten., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Isovaleryl coenzyme A (IV-CoA) performs a crucial role during development and fruiting-body formation in myxobacteria, which is reflected in the existence of a de novo biosynthetic pathway that is highly upregulated when leucine, the common precursor of IV-CoA, is limited. The final step in de novo IV-CoA biosynthesis is catalyzed by AibC, a medium-chain dehydrogenase/reductase. Here, the crystal structure of AibC from Myxococcus xanthus refined to 2.55 Å resolution is presented. The protein adopts two different conformations in the crystal lattice, which is a consequence of partial interaction with the purification tag. Based on this structure, it is suggested that AibC most probably uses a Zn(2+)-supported catalytic mechanism in which NADPH is preferred over NADH. Taken together, this study reveals structural details of the alternative IV-CoA-producing pathway in myxobacteria, which may serve as a base for further biotechnological research and biofuel production.

A critical step in bacterial isoprenoid production is the synthesis of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) catalyzed by HMG-CoA synthase (HMGCS). In myxobacteria, this enzyme is also involved in a recently discovered alternative and acetyl-CoA-dependent isovaleryl CoA biosynthesis pathway. Here we present crystal structures of MvaS, the HMGCS from Myxococcus xanthus, in complex with CoA and acetylated active site Cys115, with the second substrate acetoacetyl CoA and with the product of the condensation reaction, 3-hydroxy-3-methylglutaryl CoA. With these structures, we show that MvaS uses the common HMGCS enzymatic mechanism and provide evidence that dimerization plays a role in the formation and stability of the active site. Overall, MvaS shows features typical of the eukaryotic HMGCS and exhibits differences from homologues from Gram-positive bacteria. This study provides insights into myxobacterial alternative isovaleryl CoA biosynthesis and thereby extends the toolbox for the biotechnological production of renewable fuel and chemicals., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Myxococcus xanthus CorSR is a two-component system responsible for maintaining the response of this bacterium to copper. In the presence of this metal it upregulates, among others, the genes encoding the multicopper oxidase CuoA and the P1B -ATPase CopA. Dissection of the periplasmic sensor domain of the histidine kinase CorS by the analysis of a series of in-frame deletion mutants generated in this portion of the protein has revealed that copper sensing requires a region of 28 residues in the N terminus and another region of nine residues in the C terminus. Point mutations at His34, His38 and His171 demonstrate that they are essential for the ability of CorS to sense copper. Furthermore, the use of a bacterial two-hybrid system has revealed dimerization between monomers of CorS even in the absence of any metal, and that copper enhances this interaction. When dimerization was tested with proteins mutated at the three essential His residues, it was observed that these proteins maintain the intrinsic dimerization ability in the absence of metal. In contrast to the wild-type protein, copper did not strengthen the interaction, corroborating that copper binding to the three His residues of CorS is required for enhancing dimerization and transmitting the signal., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.

A Myxococcus xanthus gene, MXAN3487, was identified by transposon mutagenesis to be required for the expression of mcuABC, an operon coding for part of the chaperone-usher (CU) system in this bacterium. The MXAN3487 protein displays sequence and structural homology to adenosine 5'-phosphosulphate (APS) kinase family members and contains putative motifs for ATP and APS binding. Although the MXAN3487 locus is not linked to other sulphate assimilation genes, its protein product may have APS kinase activity in vivo and the importance of the ATP-binding site for activity was demonstrated. Expression of MXAN3487 was not affected by sulphate availability, suggesting that MXAN3487 may not function in a reductive sulphate assimilation pathway. Deletion of MXAN3487 significantly delayed fruiting body formation and the production of McuA, a spore coat protein secreted by the M. xanthus Mcu CU system. Based on these observations and data from our previous studies, we propose that MXAN3487 may phosphorylate molecules structurally related to APS, generating metabolites necessary for M. xanthus development, and that MXAN3487 exerts a positive effect on the mcuABC operon whose expression is morphogenesis dependent.

The site-specific conjugation of polymers to multiple engineered cysteine residues of a prolyl endopeptidase leads to its stabilization in the gastrointestinal tract of rats, without compromising the activity relative to the native enzyme. The importance of polymer attachment sites is investigated, as well as the significance of polymer structure., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Unlabelled: NADP(+) is a vital cofactor involved in a wide variety of activities, such as redox potential and cell death. Here, we show that NADP(+) negatively regulates an acetyltransferase from Myxococcus xanthus, Mxan_3215 (MxKat), at physiologic concentrations. MxKat possesses an NAD(P)-binding domain fused to the Gcn5-type N-acetyltransferase (GNAT) domain. We used isothermal titration calorimetry (ITC) and a coupled enzyme assay to show that NADP(+) bound to MxKat and that the binding had strong effects on enzyme activity. The Gly11 residue of MxKat was confirmed to play an important role in NADP(+) binding using site-directed mutagenesis and circular dichroism spectrometry. In addition, using mass spectrometry, site-directed mutagenesis, and a coupling enzymatic assay, we demonstrated that MxKat acetylates acetyl coenzyme A (acetyl-CoA) synthetase (Mxan_2570) at Lys622 in response to changes in NADP(+) concentration. Collectively, our results uncovered a mechanism of protein acetyltransferase regulation by the coenzyme NADP(+) at physiological concentrations, suggesting a novel signaling pathway for the regulation of cellular protein acetylation., Importance: Microorganisms have developed various protein posttranslational modifications (PTMs), which enable cells to respond quickly to changes in the intracellular and extracellular milieus. This work provides the first biochemical characterization of a protein acetyltransferase (MxKat) that contains a fusion between a GNAT domain and NADP(+)-binding domain with Rossmann folds, and it demonstrates a novel signaling pathway for regulating cellular protein acetylation in M. xanthus. We found that NADP(+) specifically binds to the Rossmann fold of MxKat and negatively regulates its acetyltransferase activity. This finding provides novel insight for connecting cellular metabolic status (NADP(+) metabolism) with levels of protein acetylation, and it extends our understanding of the regulatory mechanisms underlying PTMs., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Unlabelled: Escherichia coli RtcB exemplifies a family of GTP-dependent RNA repair/splicing enzymes that join 3'-PO4 ends to 5'-OH ends via stable RtcB-(histidinyl-N)-GMP and transient RNA3'pp5'G intermediates. E. coli RtcB also transfers GMP to a DNA 3'-PO4 end to form a stable "capped" product, DNA3'pp5'G. RtcB homologs are found in a multitude of bacterial proteomes, and many bacteria have genes encoding two or more RtcB paralogs; an extreme example is Myxococcus xanthus, which has six RtcBs. In this study, we purified, characterized, and compared the biochemical activities of three M. xanthus RtcB paralogs. We found that M. xanthus RtcB1 resembles E. coli RtcB in its ability to perform intra- and intermolecular sealing of a HORNAp substrate and capping of a DNA 3'-PO4 end. M. xanthus RtcB2 can splice HORNAp but has 5-fold-lower RNA ligase specific activity than RtcB1. In contrast, M. xanthus RtcB3 is distinctively feeble at ligating the HORNAp substrate, although it readily caps a DNA 3'-PO4 end. The novelty of M. xanthus RtcB3 is its capacity to cap DNA and RNA 5'-PO4 ends to form GppDNA and GppRNA products, respectively. As such, RtcB3 joins a growing list of enzymes (including RNA 3'-phosphate cyclase RtcA and thermophilic ATP-dependent RNA ligases) that can cap either end of a polynucleotide substrate. GppDNA formed by RtcB3 can be decapped to pDNA by the DNA repair enzyme aprataxin., Importance: RtcB enzymes comprise a widely distributed family of RNA 3'-PO4 ligases distinguished by their formation of 3'-GMP-capped RNAppG and/or DNAppG polynucleotides. The mechanism and biochemical repertoire of E. coli RtcB are well studied, but it is unclear whether its properties apply to the many bacteria that have genes encoding multiple RtcB paralogs. A comparison of the biochemical activities of three M. xanthus paralogs, RtcB1, RtcB2, and RtcB3, shows that not all RtcBs are created equal. The standout findings concern RtcB3, which is (i) inactive as an RNA 3'-PO4 ligase but adept at capping a DNA 3'-PO4 end and (ii) able to cap DNA and RNA 5'-PO4 ends to form GppDNA and GppRNA, respectively. The GppDNA and GppRNA capping reactions are novel nucleic acid modifications., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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., (© The Authors 2015. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.)

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., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Persulfide dioxygenases (PDOs), also known as sulfur dioxygenases (SDOs), oxidize glutathione persulfide (GSSH) to sulfite and GSH. PDOs belong to the metallo-β-lactamase superfamily and play critical roles in animals, plants, and microorganisms, including sulfide detoxification. The structures of two PDOs from human and Arabidopsis thaliana have been reported; however, little is known about the substrate binding and catalytic mechanism. The crystal structures of two bacterial PDOs from Pseudomonas putida and Myxococcus xanthus were determined at 1.5- and 2.5-Å resolution, respectively. The structures of both PDOs were homodimers, and their metal centers and β-lactamase folds were superimposable with those of related enzymes, especially the glyoxalases II. The PDOs share similar Fe(II) coordination and a secondary coordination sphere-based hydrogen bond network that is absent in glyoxalases II, in which the corresponding residues are involved instead in coordinating a second metal ion. The crystal structure of the complex between the Pseudomonas PDO and GSH also reveals the similarity of substrate binding between it and glyoxalases II. Further analysis implicates an identical mode of substrate binding by known PDOs. Thus, the data not only reveal the differences in metal binding and coordination between the dioxygenases and the hydrolytic enzymes in the metallo-β-lactamase superfamily, but also provide detailed information on substrate binding by PDOs., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Unlabelled: Gene clusters coding for the chaperone/usher (CU) pathway are widely distributed in many important environmental and pathogenic microbes; however, information about the regulatory machineries controlling CU gene expression during multicellular morphogenesis is missing. The Myxococcus xanthus Mcu system, encoded by the mcuABCD gene cluster, represents a prototype of the archaic CU family that functions in spore coat formation. Using genome-wide transposon mutagenesis, we identified MXAN2872 to be a potential regulator of the mcuABC operon and demonstrated the necessity of MXAN2872 for mcuABC expression and fruiting body morphogenesis in early development. In silico, biochemical, and genetic analyses suggest that MXAN2872 encodes a Baeyer-Villiger monooxygenase (BVMO) of flavoproteins, and the potential cofactor-binding site as well as the BVMO fingerprint sequence is important for the regulatory role of the MXAN2872 protein. The expression profile of mcuA in strains with an MXAN2872 deletion and point mutation agrees well with the timing of cell aggregation of these mutants. Furthermore, McuA could not be detected either in a fruA-null mutant, where starvation-induced aggregation was completely blocked, or in the glycerol-induced spores, where sporulation was uncoupled from cell aggregation. In sum, the present work uncovers a positive role for MXAN2872, a metabolic enzyme-encoding gene, in controlling M. xanthus development. MXAN2872 functions by affecting the onset of cell aggregation, thereby leading to a secondary effect on the timing of mcuABC expression of this model organism., Importance: Identification of the players that drive Myxococcus xanthus fruiting body formation is necessary for studying the mechanism of multicellular morphogenesis in this model organism. This study identifies MXAN2872, a gene encoding a putative flavin adenine dinucleotide-binding monooxygenase, to be a new interesting regulator regulating the timing of developmental aggregation. In addition, MXAN2872 seems to affect the expression of the chaperone/usher gene cluster mcu in a manner that is aggregation dependent. Thus, in organisms characterized by a developmental cycle, expression of the chaperone/usher pathway can be controlled by morphological checkpoints, demonstrating another layer of complexity in the regulation of this conserved protein secretion pathway., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Subunit-subunit interactions of the classical and alternate chaperone-usher (CU) systems have been shown to proceed through a donor strand exchange (DSE) mechanism. However, it is not known whether DSE is required for intersubunit interactions in the archaic CU system. We have previously shown that the Myxococcus xanthus Mcu system, a member of the archaic CU family that functions in spore coat formation, is likely to use the principle of donor strand complementation to medicate chaperone-subunit interactions analogous to the classical CU pathway. Here we describe the results of studies on Mcu subunit-subunit interactions. We constructed a series of N-terminal-deleted, single amino acid-mutated and donor strand-complemented Mcu subunits, and characterized their abilities to participate in subunit-subunit interactions. It appears that certain residues in both the N and C termini of McuA, a subunit of the Mcu system, play a critical role in intersubunit interactions and these interactions may involve the general principle of DSE of the classical and alternate CU systems. In addition, the specificity of the M. xanthus CU system for Mcu subunits over other spore coat proteins is demonstrated., (© 2014 The Authors.)

CdnL and CarD are two functionally distinct members of the CarD_CdnL_TRCF family of bacterial RNA polymerase (RNAP)-interacting proteins, which co-exist in Myxococcus xanthus. While CarD, found exclusively in myxobacteria, has been implicated in the activity of various extracytoplasmic function (ECF) σ-factors, the function and mode of action of the essential CdnL, whose homologs are widespread among bacteria, remain to be elucidated in M. xanthus. Here, we report the NMR solution structure of CdnL and present a structure-based mutational analysis of its function. An N-terminal five-stranded β-sheet Tudor-like module in the two-domain CdnL mediates binding to RNAP-β, and mutations that disrupt this interaction impair cell growth. The compact CdnL C-terminal domain consists of five α-helices folded as in some tetratricopeptide repeat-like protein-protein interaction domains, and contains a patch of solvent-exposed nonpolar and basic residues, among which a set of basic residues is shown to be crucial for CdnL function. We show that CdnL, but not its loss-of-function mutants, stabilizes formation of transcriptionally competent, open complexes by the primary σA-RNAP holoenzyme at an rRNA promoter in vitro. Consistent with this, CdnL is present at rRNA promoters in vivo. Implication of CdnL in RNAP-σA activity and of CarD in ECF-σ function in M. xanthus exemplifies how two related members within a widespread bacterial protein family have evolved to enable distinct σ-dependent promoter activity.

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., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Prolyl endopeptidases (PEP) (EC 3.4.21.26), a family of serine proteases with the ability to hydrolyze the peptide bond on the carboxyl side of an internal proline residue, are able to degrade immunotoxic peptides responsible for celiac disease (CD), such as a 33-residue gluten peptide (33-mer). Oral administration of PEP has been suggested as a potential therapeutic approach for CD, although delivery of the enzyme to the small intestine requires intrinsic gastric stability or advanced formulation technologies. We have engineered two food-grade Lactobacillus casei strains to deliver PEP in an in vitro model of small intestine environment. One strain secretes PEP into the extracellular medium, whereas the other retains PEP in the intracellular environment. The strain that secretes PEP into the extracellular medium is the most effective to degrade the 33-mer and is resistant to simulated gastrointestinal stress. Our results suggest that in the future, after more studies and clinical trials, an engineered food-grade Lactobacillus strain may be useful as a vector for in situ production of PEP in the upper small intestine of CD patients.

Myxococcus xanthus MXAN_0575, ArsA, exhibited sequence homology to low-molecular-weight protein tyrosine phosphatases (LMWPTPs) and arsenate reductases. ArsA exhibited weak phosphatase activity toward p-nitrophenyl phosphate, and high arsenate reductase activity, suggesting that ArsA may play a role in arsenate reductase, but not LMWPTP., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Fatty acid-derived ether lipids are present not only in most vertebrates but also in some bacteria. Here we describe what is to our knowledge the first gene cluster involved in the biosynthesis of such lipids in myxobacteria that encodes the multifunctional enzyme ElbD, which shows similarity to polyketide synthases. Initial characterization of elbD mutants in Myxococcus xanthus and Stigmatella aurantiaca showed the importance of these ether lipids for fruiting body formation and sporulation.