Metal-free ribonucleotide reduction powered by a DOPA radical in Mycoplasma pathogens.

Ribonucleotide reductase (RNR) catalyses the only known de novo pathway for the production of all four deoxyribonucleotides that are required for DNA synthesis1,2. It is essential for all organisms that use DNA as their genetic material and is a current drug target3,4. Since the discovery that iron is required for function in the aerobic, class I RNR found in all eukaryotes and many bacteria, a dinuclear metal site has been viewed as necessary to generate and stabilize the catalytic radical that is essential for RNR activity5-7. Here we describe a group of RNR proteins in Mollicutes-including Mycoplasma pathogens-that possess a metal-independent stable radical residing on a modified tyrosyl residue. Structural, biochemical and spectroscopic characterization reveal a stable 3,4-dihydroxyphenylalanine (DOPA) radical species that directly supports ribonucleotide reduction in vitro and in vivo. This observation overturns the presumed requirement for a dinuclear metal site in aerobic ribonucleotide reductase. The metal-independent radical requires new mechanisms for radical generation and stabilization, processes that are targeted by RNR inhibitors. It is possible that this RNR variant provides an advantage under metal starvation induced by the immune system. Organisms that encode this type of RNR-some of which are developing resistance to antibiotics-are involved in diseases of the respiratory, urinary and genital tracts. Further characterization of this RNR family and its mechanism of cofactor generation will provide insight into new enzymatic chemistry and be of value in devising strategies to combat the pathogens that utilize it. We propose that this RNR subclass is denoted class Ie.

Crystal Structures and Catalytic Mechanism of l-erythro-3,5-Diaminohexanoate Dehydrogenase and Rational Engineering for Asymmetric Synthesis of ß-Amino Acids.

Amino acid dehydrogenases (AADHs) have shown considerable potential as biocatalysts in the asymmetric synthesis of chiral amino acids. However, compared to the widely studied α-AADHs, limited knowledge is available about ß-AADHs that enable the synthesis of ß-amino acids. Herein, we report the crystal structures of a l-erythro-3,5-diaminohexanoate dehydrogenase and its variants, the only known member of ß-AADH family. Crystal structure analysis, site-directed mutagenesis studies and quantum chemical calculations revealed the differences in the substrate binding and catalytic mechanism from α-AADHs. A number of rationally engineered variants were then obtained with improved activity (by 110-800 times) toward various aliphatic ß-amino acids without an enantioselectivity trade-off. Two ß-amino acids were prepared by using the outstanding variants with excellent enantioselectivity (>99 % ee) and high isolated yields (86-87 %). These results provide important insights into the molecular mechanism of 3,5-DAHDH, and establish a solid foundation for further design of ß-AADHs for the asymmetric synthesis of ß-amino acids.

Sialidase and N-acetylneuraminate catabolism in nutrition of Mycoplasma alligatoris.

The contribution of N-acetylneuraminate scavenging to the nutrition of Mycoplasma alligatoris was examined. The wild-type grew substantially faster (P<0.01) than the mutant strains that were unable either to liberate (extracellular NanI- mutants) or to catabolize (NanA- mutants) N-acetylneuraminate from glycoconjugates in minimal SP-4 medium supplemented only with serum, but the growth of sialidase-negative mutants could not be restored to wild-type rate simply by adding unconjugated sialic acid to the culture medium. In 1 : 1 growth competition assays the wild-type was recovered in >99-fold excess of a sialidase-negative mutant after co-culture on pulmonary fibroblasts in serum-free RPMI 1640 medium, even with supplemental glucose. The advantage of nutrient scavenging via this mechanism in a complex glycan-rich environment may help to balance the expected selective disadvantage conferred by the pathogenic effects of mycoplasmal sialidase in an infected host.

Proteases as Secreted Exoproteins in Mycoplasmas from Ruminant Lungs and Their Impact on Surface-Exposed Proteins.

Many mycoplasma species are isolated from the ruminant lungs as either saprophytes or true pathogens. These wall-less bacteria possess a minimal genome and reduced metabolic capabilities. Accordingly, they rely heavily on their hosts for the supply of essential metabolites and, notably, peptides. Seven of 13 ruminant lung-associated Mycoplasma (sub)species were shown to possess caseinolytic activity when grown in rich media and assessed with a quantitative fluorescence test. For some species, this activity was detected in spent medium, an indication that proteases were secreted outside the mycoplasma cells. To identify these proteases, we incubated concentrated washed cell pellets in a defined medium and analyzed the supernatants by tandem mass spectrometry. Secreted-protease activity was detected mostly in the species belonging to the Mycoplasma mycoides cluster (MMC) and, to a lesser extent, in Mycoplasma bovirhinis Analyzing a Mycoplasma mycoides subsp. capri strain, chosen as a model, we identified 35 expressed proteases among 55 predicted coding genes, of which 5 were preferentially found in the supernatant. Serine protease S41, acquired by horizontal gene transfer, was responsible for the caseinolytic activity, as demonstrated by zymography and mutant analysis. In an M. capricolum mutant, inactivation of the S41 protease resulted in marked modification of the expression or secretion of 17 predicted surface-exposed proteins. This is an indication that the S41 protease could have a role in posttranslational cleavage of surface-exposed proteins and ectodomain shedding, whose physiological impacts still need to be explored.IMPORTANCE Few studies pertaining to proteases in ruminant mycoplasmas have been reported. Here, we focus on proteases that are secreted outside the mycoplasma cell using a mass spectrometry approach. The most striking result is the identification, within the Mycoplasma mycoides cluster, of a serine protease that is exclusively detected outside the mycoplasma cells and is responsible for casein digestion. This protease may also be involved in the posttranslational processing of surface proteins, as suggested by analysis of mutants showing a marked reduction in the secretion of extracellular proteins. By analogy, this finding may help increase understanding of the mechanisms underlying this ectodomain shedding in other mycoplasma species. The gene encoding this protease is likely to have been acquired via horizontal gene transfer from Gram-positive bacteria and sortase-associated surface proteases. Whether this protease and the associated ectodomain shedding are related to virulence has yet to be ascertained.

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.

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.

Leucine-specific domain modulates the aminoacylation and proofreading functional cycle of bacterial leucyl-tRNA synthetase.

The leucine-specific domain (LSD) is a compact well-ordered module that participates in positioning of the conserved KMSKS catalytic loop in most leucyl-tRNA synthetases (LeuRSs). However, the LeuRS from Mycoplasma mobile (MmLeuRS) has a tetrapeptide GKDG instead of the LSD. Here, we show that the tetrapeptide GKDG can confer tRNA charging and post-transfer editing activity when transplanted into an inactive Escherichia coli LeuRS (EcLeuRS) that has had its LSD deleted. Reciprocally, the LSD, together with the CP1-editing domain of EcLeuRS, can cooperate when inserted into the scaffold of the minimal MmLeuRS, and this generates an enzyme nearly as active as EcLeuRS. Further, we show that LSD participates in tRNA(Leu) recognition and favours the binding of tRNAs harbouring a large loop in the variable arm. Additional analysis established that the Lys598 in the LSD is the critical residue for tRNA binding. Conversion of Lys598 to Ala simultaneously reduces the tRNA-binding strength and aminoacylation and editing capacities, indicating that these factors are subtly connected and controlled at the level of the LSD. The present work provides a novel framework of co-evolution between LeuRS and its cognate tRNA through LSD.

Decoding system for the AUA codon by tRNAIle with the UAU anticodon in Mycoplasma mobile.

Deciphering the genetic code is a fundamental process in all living organisms. In many bacteria, AUA codons are deciphered by tRNA(Ile2) bearing lysidine (L) at the wobble position. L is a modified cytidine introduced post-transcriptionally by tRNA(Ile)-lysidine synthetase (TilS). Some bacteria, including Mycoplasma mobile, do not carry the tilS gene, indicating that they have established a different system to decode AUA codons. In this study, tRNA(Ile2) has been isolated from M. mobile and was found to contain a UAU anticodon without any modification. Mycoplasma mobile isoleucyl-tRNA synthetase (IleRS) recognized the UAU anticodon, whereas Escherichia coli IleRS did not efficiently aminoacylate tRNA(Ile2)(UAU). In M. mobile IleRS, a single Arg residue at position 865 was critical for specificity for the UAU anticodon and, when the corresponding site (W905) in E. coli IleRS was substituted with Arg, the W905R mutant efficiently aminoacylated tRNA with UAU anticodon. Mycoplasma mobile tRNA(Ile2) cannot distinguish between AUA and AUG codon on E. coli ribosome. However, on M. mobile ribosome, M. mobile tRNA(Ile2)(UAU) specifically recognized AUA codon, and not AUG codon, suggesting M. mobile ribosome has a property that prevents misreading of AUG codon. These findings provide an insight into the evolutionary reorganization of the AUA decoding system.

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.

Coordination of tRNA synthetase active sites for chemical fidelity.

Statistical proteomes that are naturally occurring can result from mechanisms involving aminoacyl-tRNA synthetases (aaRSs) with inactivated hydrolytic editing active sites. In one case, Mycoplasma mobile leucyl-tRNA synthetase (LeuRS) is uniquely missing its entire amino acid editing domain, called CP1, which is otherwise present in all known LeuRSs and also isoleucyl- and valyl-tRNA synthetases. This hydrolytic CP1 domain was fused to a synthetic core composed of a Rossmann ATP-binding fold. The fusion event splits the primary structure of the Rossmann fold into two halves. Hybrid LeuRS chimeras using M. mobile LeuRS as a scaffold were constructed to investigate the evolutionary protein:protein fusion of the CP1 editing domain to the Rossmann fold domain that is ubiquitously found in kinases and dehydrogenases, in addition to class I aaRSs. Significantly, these results determined that the modular construction of aaRSs and their adaptation to accommodate more stringent amino acid specificities included CP1-dependent distal effects on amino acid discrimination in the synthetic core. As increasingly sophisticated protein synthesis machinery evolved, the addition of the CP1 domain increased specificity in the synthetic site, as well as provided a hydrolytic editing site.

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.

Characterizing the amino acid activation center of the naturally editing-deficient aminoacyl-tRNA synthetase PheRS in Mycoplasma mobile.

To ensure that correct amino acids are incorporated during protein synthesis, aminoacyl-tRNA synthetases (aaRSs) use proofreading mechanisms collectively referred to as editing. Although editing is important for viability, editing-deficient aaRSs have been identified in host-dependent organisms. In Mycoplasma mobile, editing-deficient PheRS and LeuRS have been identified. We characterized the amino acid activation site of MmPheRS and identified a previously unknown hyperaccurate mutation, L287F. Additionally, we report that m-Tyr, an oxidation byproduct of Phe which is toxic to editing-deficient cells, is poorly discriminated by MmPheRS activation and is not subjected to editing. Furthermore, expressing MmPheRS and the hyperaccurate variants renders Escherichia coli susceptible to m-Tyr stress, indicating that active site discrimination is insufficient in tolerating excess m-Tyr.

Validation of nested PCR and a selective biochemical method as alternatives for mycoplasma detection.

Direct culture is the most common way to reliably detect mycoplasma, but it is not practical for the qualitative control of cell therapeutics because of the elaborate culture medium, the prolonged incubation time, and the large sample volumes. Here, we chose two alternative methods using commercial detection kits, the PCR mycoplasma detection kit with nested PCR and the selective biochemical method, MycoAlert(®), and validated them with the direct culture method as a reference. We tested eight mycoplasma species and five validation parameters: specificity, detection limit, robustness, repeatability, and ruggedness, based on the regulatory guidelines in the US Pharmacopoeia. All experiments were performed using fibroblasts spiked with mycoplasma. Specificity tests for both methods included all mycoplasma species, except Mycoplasma pneumonia and M. genitalium for the nested PCR and Ureaplasma urealyticum for the MycoAlert(®) assay. Regarding the detection limit, the nested PCR proved to be as sensitive as the direct culture method and more sensitive than the MycoAlert(®) assay. The predicted median for probit = 0.9 was 54 (44-76) CFU/ml for M. hyorhinis and 16 (13-23) CFU/ml for M. hominis by the nested PCR, but 431 (346-593) CFU/ml and 105 (87-142) CFU/ml, respectively, with MycoAlert(®). Changes in the concentration of reagents, reagent lot, or individual analysts did not influence the results of the examined methods. The results of this study support nested PCR as a valuable alternative for mycoplasma detection.

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.

Studies of PPLO infection. V. Inhibition of lymphocyte mitosis and antibody formation by mycoplasmal extracts.

Extracts of five arginine-utilizing mycoplasmas inhibit PHA-induced lymphocyte mitosis, while extracts of five glucose-utilizing mycoplasmas do not. Evidence is presented supporting the view that the inhibitory factor is the enzyme arginine deiminase. This enzyme inhibits the reactions of human lymphocytes to antigens as well as PHA, and the secondary production of antibody by rabbit lymph node fragments in vitro. Addition of enzyme to the cells several days after the initial mitotic or antigenic stimulus reduces, but does not abolish, further cellular activity. The production of serum proteins by hepatoma cells is totally unaffected by the mycoplasmal extract. It is concluded that arginine is an essential amino acid for the small lymphocyte, but not for the transformed cell nor for a number of other cell types. Suggestive evidence has been obtained that other enzymes similarly affect lymphocyte reactions.

Structural basis of a ribozyme's thermostability: P1-L9 interdomain interaction in RNase P RNA.

For stability, many catalytic RNAs rely on long-range tertiary interactions, the precise role of each often being unclear. Here we demonstrate that one of the three interdomain architectural struts of RNase P RNA (P RNA) is the key to activity at higher temperatures: disrupting the P1-L9 helix-tetraloop interaction in P RNA of the thermophile Thermus thermophilus decreased activity at high temperatures in the RNA-alone reaction and at low Mg2+ concentrations in the holoenzyme reaction. Conversely, implanting the P1-P9 module of T. thermophilus in the P RNA from the mesophile Escherichia coli converted the latter RNA into a thermostable one. Moreover, replacing the E. coli P1-P9 elements with a pseudoknot module that mediates the homologous interaction in Mycoplasma P RNAs not only conferred thermostability upon E. coli P RNA but also increased its maximum turnover rate at 55 degrees C to the highest yet described for a P RNA ribozyme.

Inorganic pyrophosphatase in uncultivable hemotrophic mycoplasmas: identification and properties of the enzyme from Mycoplasma suis.

BACKGROUND: Mycoplasma suis belongs to a group of highly specialized hemotrophic bacteria that attach to the surface of host erythrocytes. Hemotrophic mycoplasmas are uncultivable and the genomes are not sequenced so far. Therefore, there is a need for the clarification of essential metabolic pathways which could be crucial barriers for the establishment of an in vitro cultivation system for these veterinary significant bacteria.Inorganic pyrophosphatases (PPase) are important enzymes that catalyze the hydrolysis of inorganic pyrophosphate PPi to inorganic phosphate Pi. PPases are essential and ubiquitous metal-dependent enzymes providing a thermodynamic pull for many biosynthetic reactions. Here, we describe the identification, recombinant production and characterization of the soluble (s)PPase of Mycoplasma suis. RESULTS: Screening of genomic M. suis libraries was used to identify a gene encoding the M. suis inorganic pyrophosphatase (sPPase). The M. suis sPPase consists of 164 amino acids with a molecular mass of 20 kDa. The highest identity of 63.7% was found to the M. penetrans sPPase. The typical 13 active site residues as well as the cation binding signature could be also identified in the M. suis sPPase. The activity of the M. suis enzyme was strongly dependent on Mg2+ and significantly lower in the presence of Mn2+ and Zn2+. Addition of Ca2+ and EDTA inhibited the M. suis sPPase activity. These characteristics confirmed the affiliation of the M. suis PPase to family I soluble PPases. The highest activity was determined at pH 9.0. In M. suis the sPPase builds tetramers of 80 kDa which were detected by convalescent sera from experimentally M. suis infected pigs. CONCLUSION: The identification and characterization of the sPPase of M. suis is an additional step towards the clarification of the metabolism of hemotrophic mycoplasmas and, thus, important for the establishment of an in vitro cultivation system. As an antigenic and conserved protein the M. suis sPPase could in future be further analyzed as a diagnostic antigen.

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.

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.