Chained Structure of Dimeric F1-like ATPase in Mycoplasma mobile Gliding Machinery.

Mycoplasma mobile, a fish pathogen, exhibits gliding motility using ATP hydrolysis on solid surfaces, including animal cells. The gliding machinery can be divided into surface and internal structures. The internal structure of the motor is composed of 28 so-called "chains" that are each composed of 17 repeating protein units called "particles." These proteins include homologs of the catalytic α and ß subunits of F1-ATPase. In this study, we isolated the particles and determined their structures using negative-staining electron microscopy and high-speed atomic force microscopy. The isolated particles were composed of five proteins, MMOB1660 (α-subunit homolog), -1670 (ß-subunit homolog), -1630, -1620, and -4530, and showed ATP hydrolyzing activity. The two-dimensional (2D) structure, with dimensions of 35 and 26 nm, showed a dimer of hexameric ring approximately 12 nm in diameter, resembling F1-ATPase catalytic (αß)3. We isolated the F1-like ATPase unit, which is composed of MMOB1660, -1670, and -1630. Furthermore, we isolated the chain and analyzed the three-dimensional (3D) structure, showing that dimers of mushroom-like structures resembling F1-ATPase were connected and aligned along the dimer axis at 31-nm intervals. An atomic model of F1-ATPase catalytic (αß)3 from Bacillus PS3 was successfully fitted to each hexameric ring of the mushroom-like structure. These results suggest that the motor for M. mobile gliding shares an evolutionary origin with F1-ATPase. Based on the obtained structure, we propose possible force transmission processes in the gliding mechanism. IMPORTANCE F1Fo-ATPase, a rotary ATPase, is widespread in the membranes of mitochondria, chloroplasts, and bacteria and converts ATP energy with a proton motive force across the membrane by its physical rotation. Homologous protein complexes play roles in ion and protein transport. Mycoplasma mobile, a pathogenic bacterium, was recently suggested to have a special motility system evolutionarily derived from F1-ATPase. The present study isolated the protein complex from Mycoplasma cells and supported this conclusion by clarifying the detailed structures containing common and novel features as F1-ATPase relatives.

Refined Mechanism of Mycoplasma mobile Gliding Based on Structure, ATPase Activity, and Sialic Acid Binding of Machinery.

Mycoplasma mobile, a fish pathogen, glides on solid surfaces by repeated catch, pull, and release of sialylated oligosaccharides by a unique mechanism based on ATP energy. The gliding machinery is composed of huge surface proteins and an internal "jellyfish"-like structure. Here, we elucidated the detailed three-dimensional structures of the machinery by electron cryotomography. The internal "tentacle"-like structure hydrolyzed ATP, which was consistent with the fact that the paralogs of the α- and ß-subunits of F1-ATPase are at the tentacle structure. The electron microscopy suggested conformational changes of the tentacle structure depending on the presence of ATP analogs. The gliding machinery was isolated and showed that the binding activity to sialylated oligosaccharide was higher in the presence of ADP than in the presence of ATP. Based on these results, we proposed a model to explain the mechanism of M. mobile gliding.IMPORTANCE The genus Mycoplasma is made up of the smallest parasitic and sometimes commensal bacteria; Mycoplasma pneumoniae, which causes human "walking pneumonia," is representative. More than ten Mycoplasma species glide on host tissues by novel mechanisms, always in the direction of the distal side of the machinery. Mycoplasma mobile, the fastest species in the genus, catches, pulls, and releases sialylated oligosaccharides (SOs), the carbohydrate molecules also targeted by influenza viruses, by means of a specific receptor and using ATP hydrolysis for energy. Here, the architecture of the gliding machinery was visualized three dimensionally by electron cryotomography (ECT), and changes in the structure and binding activity coupled to ATP hydrolysis were discovered. Based on the results, a refined mechanism was proposed for this unique motility.

Arginine Deiminase: Current Understanding and Applications.

BACKGROUND: Arginine deiminase (ADI), an arginine catabolizing enzyme, is considered as an anti-tumor agent for the treatment of arginine auxotrophic cancers. However, some obstacles limit its clinical applications. OBJECTIVE: This review will summarize the clinical applications of ADI, from a brief history to its limitations, and will discuss the different ways to deal with the clinical limitations. METHOD: The structure analysis, cloning, expression, protein engineering and applications of arginine deiminase enzyme have been explained in this review. CONCLUSION: Recent patents on ADI are related to ADI engineering to increase its efficacy for clinical application. The intracellular delivery of ADI and combination therapy seem to be the future strategies in the treatment of arginine auxotrophic cancers. Applying ADIs with optimum features from different sources and or ADI engineering, are promising strategies to improve the clinical application of ADI.

Arginine Deiminase Enzyme Evolving as a Potential Antitumor Agent.

Some melanomas and hepatocellular carcinomas have been shown to be auxotrophic for arginine. Arginine deiminase (ADI), an arginine degrading enzyme isolated from Mycoplasma, can inhibit the growth of these tumors. It is a catabolizing enzyme which catabolizes arginine to Citrulline. Tumor cells do not express an enzyme called arginosuccinate synthetase (ASS) and hence, these cells become auxotrophic for arginine. It is found that ADI is specific for arginine and did not degrade other amino acid. This review covers various aspects of ADIs like origin, properties and chemical modifications for better antitumor activity.

Arginine deiminase: recent advances in discovery, crystal structure, and protein engineering for improved properties as an anti-tumor drug.

Arginine deiminase (ADI) is an important arginine-degrading enzyme with wide applications, in particular as an anti-cancer agent for the therapy of arginine-auxotrophic tumors. In recent years, novel ADIs with excellent properties have been identified from various organisms, and crystal structures of ADI were investigated. To satisfy the requirements of potential therapeutic applications, protein engineering has been performed to improve the activity and properties of ADIs. In this mini-review, we systematically summarized the latest progress on identification and crystal structure of ADIs, and protein engineering strategies for improved enzymatic properties, such as pH optimum, K m and k cat values, and thermostability. We also outlined the PEGylation of ADI for improved circulating half-life and immunogenicity, as well as their performance in clinical trials. Finally, perspectives on extracellular secretion and property improvement of ADI were discussed.

Modulation of Aminoacylation and Editing Properties of Leucyl-tRNA Synthetase by a Conserved Structural Module.

A conserved structural module following the KMSKS catalytic loop exhibits α-α-ß-α topology in class Ia and Ib aminoacyl-tRNA synthetases. However, the function of this domain has received little attention. Here, we describe the effect this module has on the aminoacylation and editing capacities of leucyl-tRNA synthetases (LeuRSs) by characterizing the key residues from various species. Mutation of highly conserved basic residues on the third α-helix of this domain impairs the affinity of LeuRS for the anticodon stem of tRNA(Leu), which decreases both aminoacylation and editing activities. Two glycine residues on this α-helix contribute to flexibility, leucine activation, and editing of LeuRS from Escherichia coli (EcLeuRS). Acidic residues on the ß-strand enhance the editing activity of EcLeuRS and sense the size of the tRNA(Leu) D-loop. Incorporation of these residues stimulates the tRNA-dependent editing activity of the chimeric minimalist enzyme Mycoplasma mobile LeuRS fused to the connective polypeptide 1 editing domain and leucine-specific domain from EcLeuRS. Together, these results reveal the stem contact-fold to be a functional as well as a structural linker between the catalytic site and the tRNA binding domain. Sequence comparison of the EcLeuRS stem contact-fold domain with editing-deficient enzymes suggests that key residues of this module have evolved an adaptive strategy to follow the editing functions of LeuRS.

Synthesis of Mycoplasma arginine deiminase in E. coli using stress-responsive proteins.

We found Escherichia coli proteins, elongation factor Ts (Tsf), and malate dehydrogenase (Mdh) that can exist in the form of native and soluble proteins even under stress situation such as heat shock and protein denaturing condition. To examine their property as solubility enhancers, aggregation-prone Mycoplasma arginine deiminase (mADI), which has been suggested as anti-cancer agent, was fused to the C-terminal of each of them and cloned into pET28a to be expressed in the E. coli cytoplasm. When mADI was fused to fusion partners (Mdh, Tsf), a significant portion of the recombinant mADI was expressed in a soluble fraction (>90%) whereas the directly expressed mADI was aggregated to the inclusion body. In addition, recombinant mADI released from the fusion tag retained its soluble form and presented its specific enzymatic activity of converting l-arginine into citrulline (>10 U/mg). These results show that Tsf and Mdh could serve as effective solubility enhancers for aggregation-prone proteins (e.g. mADI in this case) when used as fusion expression partners in bacterial expression systems.

A small GTPase­like protein fragment of Mycoplasma promotes tumor cell migration and proliferation in vitro via interaction with Rac1 and Stat3.

The Mycoplasma genus comprises a group of microbes that cause persistent infection in humans and its role in promoting tumor development has long been a concern. Although mixtures of components isolated from Mycoplasma have been shown to activate host Rho family small GTPases and Stat3, no individual factor with this activity has been reported. In the current study, a conserved small GTPase-like protein fragment (SGLP) from Mycoplasma pulmonis chromosome partition protein, Smc, was identified as a virulence factor. SGLP was observed to interact with Rac1 and Stat3. The wild­type (wt) SGLP, which contains a WxxxE motif, induced activation of Rac1 and phosphorylation of Stat3 at the tyrosine­705 residue, while the SGLP mutant containing a mutation from WxxxE to AxxxA did not exert the same effects. Moreover, SGLP­induced Stat3 phosphorylation was observed to be dependent upon Rac1 activity. Furthermore, wt SGLP was observed to promote cell migration and increase bromodeoxyuridine incorporation in HeLa cells and the SGLP mutant did not elicit these effects in HeLa cells. In conclusion, the current observations suggest that SGLP is an important virulence factor of Mycoplasma, which contributes to tumor cell migration and proliferation in vitro via interaction with Rac1 and Stat3.

Life without tRNAArg-adenosine deaminase TadA: evolutionary consequences of decoding the four CGN codons as arginine in Mycoplasmas and other Mollicutes.

In most bacteria, two tRNAs decode the four arginine CGN codons. One tRNA harboring a wobble inosine (tRNA(Arg)ICG) reads the CGU, CGC and CGA codons, whereas a second tRNA harboring a wobble cytidine (tRNA(Arg)CCG) reads the remaining CGG codon. The reduced genomes of Mycoplasmas and other Mollicutes lack the gene encoding tRNA(Arg)CCG. This raises the question of how these organisms decode CGG codons. Examination of 36 Mollicute genomes for genes encoding tRNA(Arg) and the TadA enzyme, responsible for wobble inosine formation, suggested an evolutionary scenario where tadA gene mutations first occurred. This allowed the temporary accumulation of non-deaminated tRNA(Arg)ACG, capable of reading all CGN codons. This hypothesis was verified in Mycoplasma capricolum, which contains a small fraction of tRNA(Arg)ACG with a non-deaminated wobble adenosine. Subsets of Mollicutes continued to evolve by losing both the mutated tRNA(Arg)CCG and tadA, and then acquired a new tRNA(Arg)UCG. This permitted further tRNA(Arg)ACG mutations with tRNA(Arg)GCG or its disappearance, leaving a single tRNA(Arg)UCG to decode the four CGN codons. The key point of our model is that the A-to-I deamination activity had to be controlled before the loss of the tadA gene, allowing the stepwise evolution of Mollicutes toward an alternative decoding strategy.

Specific evolution of F1-like ATPases in mycoplasmas.

F(1)F(0) ATPases have been identified in most bacteria, including mycoplasmas which have very small genomes associated with a host-dependent lifestyle. In addition to the typical operon of eight genes encoding genuine F(1)F(0) ATPase (Type 1), we identified related clusters of seven genes in many mycoplasma species. Four of the encoded proteins have predicted structures similar to the α, ß, γ and ε subunits of F(1) ATPases and could form an F(1)-like ATPase. The other three proteins display no similarity to any other known proteins. Two of these proteins are probably located in the membrane, as they have three and twelve predicted transmembrane helices. Phylogenomic studies identified two types of F(1)-like ATPase clusters, Type 2 and Type 3, characterized by a rapid evolution of sequences with the conservation of structural features. Clusters encoding Type 2 and Type 3 ATPases were assumed to originate from the Hominis group of mycoplasmas. We suggest that Type 3 ATPase clusters may spread to other phylogenetic groups by horizontal gene transfer between mycoplasmas in the same host, based on phylogeny and genomic context. Functional analyses in the ruminant pathogen Mycoplasma mycoides subsp. mycoides showed that the Type 3 cluster genes were organized into an operon. Proteomic analyses demonstrated that the seven encoded proteins were produced during growth in axenic media. Mutagenesis and complementation studies demonstrated an association of the Type 3 cluster with a major ATPase activity of membrane fractions. Thus, despite their tendency toward genome reduction, mycoplasmas have evolved and exchanged specific F(1)-like ATPases with no known equivalent in other bacteria. We propose a model, in which the F(1)-like structure is associated with a hypothetical X(0) sector located in the membrane of mycoplasma cells.

A naturally occurring nonapeptide functionally compensates for the CP1 domain of leucyl-tRNA synthetase to modulate aminoacylation activity.

aaRSs (aminoacyl-tRNA synthetases) establish the rules of the genetic code by catalysing the formation of aminoacyl-tRNA. The quality control for aminoacylation is achieved by editing activity, which is usually carried out by a discrete editing domain. For LeuRS (leucyl-tRNA synthetase), the CP1 (connective peptide 1) domain is the editing domain responsible for hydrolysing mischarged tRNA. The CP1 domain is universally present in LeuRSs, except MmLeuRS (Mycoplasma mobile LeuRS). The substitute of CP1 in MmLeuRS is a nonapeptide (MmLinker). In the present study, we show that the MmLinker, which is critical for the aminoacylation activity of MmLeuRS, could confer remarkable tRNA-charging activity on the inactive CP1-deleted LeuRS from Escherichia coli (EcLeuRS) and Aquifex aeolicus (AaLeuRS). Furthermore, CP1 from EcLeuRS could functionally compensate for the MmLinker and endow MmLeuRS with post-transfer editing capability. These investigations provide a mechanistic framework for the modular construction of aaRSs and their co-ordination to achieve catalytic efficiency and fidelity. These results also show that the pre-transfer editing function of LeuRS originates from its conserved synthetic domain and shed light on future study of the mechanism.

Improvement of purine and pyrimidine antimetabolite-based anticancer treatment by selective suppression of mycoplasma-encoded catabolic enzymes.

Most mycoplasmas are present as commensals, colonising the mucosa of our respiratory and gastrointestinal tract. Experimental data suggest that the long-term association of certain mycoplasma species with mammalian cells might favour host-cell transformation and malignancy. Moreover, increased mycoplasma infection has been noted in several cancers. Despite efforts to develop target-specific anticancer drugs, current cancer treatment still relies on the use of nucleobase or nucleoside-based analogues. Here, we provide experimental evidence that nucleoside-metabolising catabolic enzymes expressed by mycoplasmas substantially compromise the efficacy of nucleoside antimetabolites used in the treatment of cancer. We also suggest potential methods for improving future chemotherapy by suppressing mycoplasma-mediated catabolism of the anticancer nucleoside analogues.

Arginine deiminase enhances MCF-7 cell radiosensitivity by inducing changes in the expression of cell cycle-related proteins.

After successful clinical application, arginine deiminase (ADI) has been proposed to be a new cancer therapeutic. In the present study, we examined the effect of ADI in combination with ionizing radiation (IR) on MCF-7 cell growth and clonogenic cell death. Cell growth was inhibited by IR in a dose-dependent manner and ADI enhanced the radiosensitivity. ADI itself did not suppress the growth of MCF-7 cells due to the high level of expression of argininosuccinate synthetase (ASS), which convert citrulline, a product of arginine degradation by ADI, to arginine. Previously, it was suggested that ammonia, another product of arginine degradation by ADI, is the main cause of the growth inhibition of irradiated hepatoma cells contaminated with ADI-expressing mycoplasma [van Rijn et al. (2003)]. However, we found that ammonia is not the only factor that enhances radiosensitivity, as enhancement was also observed in the absence of ammonia. In order to identify the enhancing effect, levels of ASS and proteins related to the cell cycle were examined. ASS was unchanged by ADI plus IR, but p21 (a CDK inhibitor) was upregulated and c-Myc downregulated. These findings indicate that changes in the expressions of cell cycle proteins are involved in the enhancement of radiosensitivity by ADI. We suggest that ADI is a potential adjunct to cancer therapy.

Role of sialidase in Mycoplasma alligatoris-induced pulmonary fibroblast apoptosis.

Mycoplasma alligatoris causes acute lethal cardiopulmonary disease of susceptible hosts. A survey of its genome implicated sialidase and hyaluronidase, synergistic regulators of hyaluronan receptor CD44-mediated signal transduction leading to apoptotic cell death, as virulence factors of M. alligatoris. In this study, after the existence of a CD44 homolog in alligators was established by immunolabeling primary pulmonary fibroblasts with monoclonal antibody IM7 against murine CD44, the sialidase inhibitor 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) was used to examine the effects of sialidase on fibroblast apoptosis following in vitro infection with M. alligatoris. While their CD44 expression remained constant, infected cells exhibited morphologic changes characteristic of apoptosis including decreased size, rounding, disordered alpha-tubulin, and nuclear disintegration compared to untreated controls. DANA was a potent, non-toxic inhibitor of the sialidase activity, equivalent to about 1mU of Clostridium perfringens Type VI sialidase, expressed by M. alligatoris in the inoculum. Although DANA did not measurably reduce the proportion of infected fibroblasts labeled by a specific ligand of activated caspases, co-incubation with DANA protected (P<0.01) fibroblasts in a concentration-dependent fashion from the M. alligatoris-induced trends toward increased apoptosis receptor CD95 expression, and increased 5-bromo-2'-deoxyuridine incorporation measured in a terminal dUTP nick end-labeling apoptosis assay. In contrast, incubation with 200-fold excess purified C. perfringens sialidase alone did not affect CD95 expression or chromatin integrity, or induce fibroblast apoptosis. From those observations we conclude that interaction of its sialidase with hyaluronidase or another virulence factor(s) is necessary to elicit the pro-apoptotic effects of M. alligatoris infection.

Suppression of human immunodeficiency virus type 1 replication by arginine deiminase of Mycoplasma arginini.

It was found previously that human immunodeficiency virus type 1 (HIV-1)-irrelevant CD8+ cytotoxic T lymphocytes (CTLs) from uninfected donors suppressed HIV-1 replication in a cell-contact-dependent manner. However, one of these CTL lines (CTL-3) also significantly suppressed HIV-1 replication through its supernatant. Here, the suppressive fraction from CTL-3 supernatant was purified and analysed by mass spectrometry. A protein band specific for the suppressive fraction was identified as arginine deiminase from Mycoplasma arginini, which catalyses the hydrolysis of arginine to citrulline. Addition of L-arginine or the use of antibiotics against mycoplasma restored supernatant-mediated but not cell-contact-dependent suppression of HIV-1 replication by CTL-3, clearly indicating that arginine deiminase of M. arginini in the supernatants suppressed HIV-1 replication, which is independent of CD8+ T-cell-mediated HIV-1 suppression via cell contact. Arginine deiminase is known to be a chemotherapeutic agent against arginine-requiring tumours and these results suggest that it also has potential application in antiviral therapy.

Structures of thymidine kinase 1 of human and mycoplasmic origin.

Cytosolic thymidine kinase 1, TK1, is a well known cell-cycle-regulated enzyme of importance in nucleotide metabolism as well as an activator of antiviral and anticancer drugs such as 3'-azido-3'-deoxythymidine (AZT). We have now determined the structures of the TK1 family, the human and Ureaplasma urealyticum enzymes, in complex with the feedback inhibitor dTTP. The TK1s have a tetrameric structure in which each subunit contains an alpha/beta-domain that is similar to ATPase domains of members of the RecA structural family and a domain containing a structural zinc. The zinc ion connects beta-structures at the root of a beta-ribbon that forms a stem that widens to a lasso-type loop. The thymidine of dTTP is hydrogen-bonded to main-chain atoms predominantly coming from the lasso loop. This binding is in contrast to other deoxyribonucleoside kinases where specific interactions occur with side chains. The TK1 structure differs fundamentally from the structures of the other deoxyribonucleoside kinases, indicating a different evolutionary origin.

Crystal structures of arginine deiminase with covalent reaction intermediates; implications for catalytic mechanism.

Arginine deiminase (ADI), an enzyme that hydrolyzes arginine to generate energy in many parasitic microorganisms, has potent anticancer activities and can halt growth of solid tumors. We determined the crystal structure of ADI from Mycoplasma arginini in two different forms (1.6 and 2.0 A resolution) using multiple isomorphous replacement. ADI shares common structural features with the arginine-catabolizing enzymes Arg:Gly amidinotransferase and dimethylarginine dimethyl-aminohydrolase; ADI contains an additional domain of five helices. The scissile C-N bonds of the substrates and the catalytic triads (Cys398-His269-Glu213 of ADI) for the three enzymes superimpose on each other. The ADI structure from form I crystals corresponds to a tetrahedral intermediate with four heteroatoms (1S, 2N, 1O) covalently bonded to the reaction-center carbon. The structure from form II crystals represents an amidino-enzyme complex; the reaction-center carbon is covalently bonded to Cys398 sulfur and two nitrogens, and the reacting water molecule is only 2.54 A away.

Resistance to the anti-proliferative activity of recombinant arginine deiminase in cell culture correlates with the endogenous enzyme, argininosuccinate synthetase.

Recombinant mycoplasma enzyme, arginine deiminase (rADI), has been proposed as a possible cancer treatment via arginine depletion. However, many cell lines are resistant to rADI-treatment, even though most require arginine for proliferation. We compared eight different cell lines for sensitivity in cell proliferation to the effect of either rADI or arginine deprivation. The activity of argininosuccinate synthetase (AS), the rate-limiting enzyme for converting citrulline to arginine, was also measured. Our results indicate that resistance to rADI-treatment may correlate with cellular AS activity, either constitutive or inducible, allowing cell survival by conversion of the product of the rADI reaction, i.e. citrulline to arginine.

Suspected utility of enzymes with multiple activities in the small genome Mycoplasma species: the replacement of the missing "household" nucleoside diphosphate kinase gene and activity by glycolytic kinases.

The small genome Mollicutes whose DNAs are completely sequenced (Mycoplasma genitalium, Mycoplasma pneumoniae, Mycoplasma pulmonis, and Ureaplasma urealyticum [parvum]) lack a gene (ndk) for the presumably essential nucleoside diphosphate kinase (NDPK). We hypothesized that other activities might replace NDPK activity. We found in M. genitalium G37(T), Mycoplasma pneumoniae FH(T), Mycoplasma fermentans PG18(T), and Mycoplasma capricolum subsp. capricolum Kid(T) that their 6-phosphofructokinases (6-PFKs), phosphoglycerate kinases (PGKs), pyruvate kinases (PKs), and acetate kinases (AKs), besides reactant ADP/ATP, could use other ribo- and deoxyribo-purine and pyrimidine NDPs and NTPs. These activities could compensate for the absence of an orthologous ndk gene in the Mycoplasmataceae. They suggest a metabolically varied and consequential role for unrelated and perhaps unsuspected "replacement" or compensatory enzymes that may confound metabolic prediction. We partially purified and biochemically characterized the PKs, 6-PFKs, PGKs, and AKs from M. capricolum subsp. capricolum Kid(T) and M. fermentans PG18(T).

Diagnosis and treatment of mycoplasma-contaminated cell cultures.

Mycoplasma contamination is a serious and frequent problem in the culture laboratory. Although mycoplasma contamination may be suspected by the failure of cells to thrive, the formal diagnosis rests on the detection of adenosine phosphorylase secretion by infected cell lines. This appendix describes how to test for mycoplasma contamination, and also presents methods for antibiotic treatment of infected cultures.