Zng1 is a GTP-dependent zinc transferase needed for activation of methionine aminopeptidase.

The evolution of zinc (Zn) as a protein cofactor altered the functional landscape of biology, but dependency on Zn also created an Achilles' heel, necessitating adaptive mechanisms to ensure Zn availability to proteins. A debated strategy is whether metallochaperones exist to prioritize essential Zn-dependent proteins. Here, we present evidence for a conserved family of putative metal transferases in human and fungi, which interact with Zn-dependent methionine aminopeptidase type I (MetAP1/Map1p/Fma1). Deletion of the putative metal transferase in Saccharomyces cerevisiae (ZNG1; formerly YNR029c) leads to defective Map1p function and a Zn-deficiency growth defect. In vitro, Zng1p can transfer Zn2+ or Co2+ to apo-Map1p, but unlike characterized copper chaperones, transfer is dependent on GTP hydrolysis. Proteomics reveal mis-regulation of the Zap1p transcription factor regulon because of loss of ZNG1 and Map1p activity, suggesting that Zng1p is required to avoid a compounding effect of Map1p dysfunction on survival during Zn limitation.

Characterization of a Cul9-Parkin double knockout mouse model for Parkinson's disease.

Mitochondrial quality control is essential for the long-term survival of postmitotic neurons. The E3 ubiquitin ligase Parkin promotes the degradation of damaged mitochondria via mitophagy and mutations in Parkin are a major cause of early-onset Parkinson's disease (PD). Surprisingly however, mice deleted for Parkin alone are rather asymptomatic for PD-related pathology, suggesting that other complementary or redundant mitochondrial quality control pathways may exist in neurons. Mitochondrial damage is often accompanied by the release of toxic proteins such as cytochrome c. We have reported that once in the cytosol, cytochrome c is targeted for degradation by the E3 ligase CUL9 in neurons. Here we examined whether CUL9 and Parkin cooperate to promote optimal neuronal survival in vivo. We generated mice deficient for both Cul9 and Parkin and examined them for PD-related phenotypes. Specifically, we conducted assays to examine behavioural deficits (locomotor, sensory, memory and learning) and loss of dopaminergic neurons in both males and females. Our results show that the loss of Cul9 and Parkin together did not enhance the effect of Parkin deficiency alone. These results indicate that while both Parkin and CUL9 participate in mitochondrial quality control, neurons likely have multiple redundant mechanisms to ensure their long-term survival.

The organ-specific differential roles of rice DXS and DXR, the first two enzymes of the MEP pathway, in carotenoid metabolism in Oryza sativa leaves and seeds.

BACKGROUND: Deoxyxylulose 5-phosphate synthase (DXS) and deoxyxylulose 5-phosphate reductoisomerase (DXR) are the enzymes that catalyze the first two enzyme steps of the methylerythritol 4-phosphate (MEP) pathway to supply the isoprene building-blocks of carotenoids. Plant DXR and DXS enzymes have been reported to function differently depending on the plant species. In this study, the differential roles of rice DXS and DXR genes in carotenoid metabolism were investigated. RESULTS: The accumulation of carotenoids in rice seeds co-expressing OsDXS2 and stPAC was largely enhanced by 3.4-fold relative to the stPAC seeds and 315.3-fold relative to non-transgenic (NT) seeds, while the overexpression of each OsDXS2 or OsDXR caused no positive effect on the accumulation of either carotenoids or chlorophylls in leaves and seeds, suggesting that OsDXS2 functions as a rate-limiting enzyme supplying IPP/DMAPPs to seed carotenoid metabolism, but OsDXR doesn't in either leaves or seeds. The expressions of OsDXS1, OsPSY1, OsPSY2, and OsBCH2 genes were upregulated regardless of the reductions of chlorophylls and carotenoids in leaves; however, there was no significant change in the expression of most carotenogenic genes, even though there was a 315.3-fold increase in the amount of carotenoid in rice seeds. These non-proportional expression patterns in leaves and seeds suggest that those metabolic changes of carotenoids were associated with overexpression of the OsDXS2, OsDXR and stPAC transgenes, and the capacities of the intermediate biosynthetic enzymes might be much more important for those metabolic alterations than the transcript levels of intermediate biosynthetic genes are. Taken together, we propose a 'Three Faucets and Cisterns Model' about the relationship among the rate-limiting enzymes OsDXSs, OsPSYs, and OsBCHs as a "Faucet", the biosynthetic capacity of intermediate metabolites as a "Cistern", and the carotenoid accumulations as the content of "Cistern". CONCLUSION: Our study suggests that OsDXS2 plays an important role as a rate-limiting enzyme supplying IPP/DMAPPs to the seed-carotenoid accumulation, and rice seed carotenoid metabolism could be largely enhanced without any significant transcriptional alteration of carotenogenic genes. Finally, the "Three Faucets and Cisterns model" presents the extenuating circumstance to elucidate rice seed carotenoid metabolism.

PhiA, a Peptidoglycan Hydrolase Inhibitor of Brucella Involved in the Virulence Process.

The peptidoglycan in Gram-negative bacteria is a dynamic structure in constant remodeling. This dynamism, achieved through synthesis and degradation, is essential because the peptidoglycan is necessary to maintain the structure of the cell but has to have enough plasticity to allow the transport and assembly of macromolecular complexes in the periplasm and outer membrane. In addition, this remodeling has to be coordinated with the division process. Among the multiple mechanisms bacteria have to degrade the peptidoglycan are the lytic transglycosidases, enzymes of the lysozyme family that cleave the glycan chains generating gaps in the mesh structure increasing its permeability. Because these enzymes can act as autolysins, their activity has to be tightly regulated, and one of the mechanisms bacteria have evolved is the synthesis of membrane bound or periplasmic inhibitors. In the present study, we identify a periplasmic lytic transglycosidase inhibitor (PhiA) in Brucella abortus and demonstrate that it inhibits the activity of SagA, a lytic transglycosidase we have previously shown is involved in the assembly of the type IV secretion system. A phiA deletion mutant results in a strain with the incapacity to synthesize a complete lipopolysaccharide but with a higher replication rate than the wild-type parental strain, suggesting a link between peptidoglycan remodeling and speed of multiplication.

MCR-1: a promising target for structure-based design of inhibitors to tackle polymyxin resistance.

The spread of a novel mobile colistin resistance gene (mcr1) has jeopardised the use of polymyxins, last-resort antibiotics that are used increasingly to treat infections caused by multidrug-resistant (MDR) Gram-negative pathogens. In early 2017, the WHO reported the global spread of mcr1 within a few years after its initial discovery in China. The protein encoded by mcr1 is a putative 60-kDa phosphoethanolamine (pEtN) transferase, MCR-1, and has been studied extensively since its discovery. Herein, we present a comprehensive review of MCR-1 covering its structure, function, and mechanism, to call for the rational drug design of molecular inhibitors of MCR-1 to use in colistin-based combination therapies.

Crosstalk between the lipopolysaccharide and phospholipid pathways during outer membrane biogenesis in Escherichia coli.

The outer membrane of gram-negative bacteria is composed of phospholipids in the inner leaflet and lipopolysaccharides (LPS) in the outer leaflet. LPS is an endotoxin that elicits a strong immune response from humans, and its biosynthesis is in part regulated via degradation of LpxC (EC 3.5.1.108) and WaaA (EC 2.4.99.12/13) enzymes by the protease FtsH (EC 3.4.24.-). Because the synthetic pathways for both molecules are complex, in addition to being produced in strict ratios, we developed a computational model to interrogate the regulatory mechanisms involved. Our model findings indicate that the catalytic activity of LpxK (EC 2.7.1.130) appears to be dependent on the concentration of unsaturated fatty acids. This is biologically important because it assists in maintaining LPS/phospholipids homeostasis. Further crosstalk between the phospholipid and LPS biosynthetic pathways was revealed by experimental observations that LpxC is additionally regulated by an unidentified protease whose activity is independent of lipid A disaccharide concentration (the feedback source for FtsH-mediated LpxC regulation) but could be induced in vitro by palmitic acid. Further experimental analysis provided evidence on the rationale for WaaA regulation. Overexpression of waaA resulted in increased levels of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) sugar in membrane extracts, whereas Kdo and heptose levels were not elevated in LPS. This implies that uncontrolled production of WaaA does not increase the LPS production rate but rather reglycosylates lipid A precursors. Overall, the findings of this work provide previously unidentified insights into the complex biogenesis of the Escherichia coli outer membrane.

cis-Prenyltransferase atCPT6 produces a family of very short-chain polyisoprenoids in planta.

cis-Prenyltransferases (CPTs) comprise numerous enzymes synthesizing isoprenoid hydrocarbon skeleton with isoprenoid units in the cis (Z) configuration. The chain-length specificity of a particular plant CPT is in most cases unknown despite thecomposition of the accumulated isoprenoids in the tissue of interest being well established. In this report AtCPT6, one of the nine Arabidopsis thaliana CPTs, is shown to catalyze the synthesis of a family of very short-chain polyisoprenoid alcohols of six, seven, and eight isoprenoid units, those of seven units dominating The product specificity of AtCPT6 was established in vivo following its expression in the heterologous system of the yeast Saccharomyces cerevisiae and was confirmed by the absence of specific products in AtCPT6 T-DNA insertion mutants and their overaccumulation in AtCPT6-overexpressing plants. These observations are additionally validated in silico using an AtCPT6 model obtained by homology modeling. AtCPT6 only partially complements the function of the yeast homologue of CPT-Rer2 since it restores the growth but not protein glycosylation in rer2delta yeast.This is the first in planta characterization of specific products of a plant CPT producing polyisoprenoids. Their distribution suggests that a joint activity of several CPTs is required to produce the complex mixture of polyisoprenoid alcohols found in Arabidopsis roots.

Branched signal wiring of an essential bacterial cell-cycle phosphotransfer protein.

Vital to bacterial survival is the faithful propagation of cellular signals, and in Caulobacter crescentus, ChpT is an essential mediator within the cell-cycle circuit. ChpT functions as a histidine-containing phosphotransfer protein (HPt) that shuttles a phosphoryl group from the receiver domain of CckA, the upstream hybrid histidine kinase (HK), to one of two downstream response regulators (CtrA or CpdR) that controls cell-cycle progression. To understand how ChpT interacts with multiple signaling partners, we solved the crystal structure of ChpT at 2.3 Å resolution. ChpT adopts a pseudo-HK architecture but does not bind ATP. We identified two point mutation classes affecting phosphotransfer and cell morphology: one that globally impairs ChpT phosphotransfer, and a second that mediates partner selection. Importantly, a small set of conserved ChpT residues promotes signaling crosstalk and contributes to the branched signaling that activates the master regulator CtrA while inactivating the CtrA degradation signal, CpdR.

Microbial interactions: bacteria talk to (some of) their neighbors.

A recent study reports that Bacillus subtilis biofilm formation depends upon paracrine signaling where the signal-producing and target-responsive cells are different.

WaaA of the hyperthermophilic bacterium Aquifex aeolicus is a monofunctional 3-deoxy-D-manno-oct-2-ulosonic acid transferase involved in lipopolysaccharide biosynthesis.

The hyperthermophile Aquifex aeolicus belongs to the deepest branch in the bacterial genealogy. Although it has long been recognized that this unique Gram-negative bacterium carries genes for different steps of lipopolysaccharide (LPS) formation, data on the LPS itself or detailed knowledge of the LPS pathway beyond the first committed steps of lipid A and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) synthesis are still lacking. We now report the functional characterization of the thermostable Kdo transferase WaaA from A. aeolicus and provide evidence that the enzyme is monofunctional. Compositional analysis and mass spectrometry of purified A. aeolicus LPS, showing the incorporation of a single Kdo residue as an integral component of the LPS, implicated a monofunctional Kdo transferase in LPS biosynthesis of A. aeolicus. Further, heterologous expression of the A. aeolicus waaA gene in a newly constructed Escherichia coli DeltawaaA suppressor strain resulted in synthesis of lipid IVA precursors substituted with one Kdo sugar. When highly purified WaaA of A. aeolicus was subjected to in vitro assays using mass spectrometry for detection of the reaction products, the enzyme was found to catalyze the transfer of only a single Kdo residue from CMP-Kdo to differently modified lipid A acceptors. The Kdo transferase was capable of utilizing a broad spectrum of acceptor substrates, whereas surface plasmon resonance studies indicated a high selectivity for the donor substrate.

The pyrH gene of Vibrio vulnificus is an essential in vivo survival factor.

We have suggested an important role of the pyrH gene during the infectious process of Vibrio vulnificus. Previously, we have identified 12 genes expressed preferentially during human infections by using in vivo-induced antigen technology. Among the in vivo-expressed genes, pyrH encodes UMP kinase catalyzing UMP phosphorylation. Introduction of a deletion mutation to the pyrH gene was lethal to V. vulnificus, and an insertional mutant showed a high frequency of curing. We constructed a site-directed mutant strain (R62H/D77N) on Arg-62 and Asp-77, both predicted to be involved in UMP binding, and characterized the R62H/D77N strain compared with the previously reported insertional mutant. We further investigated the essential role of the pyrH gene in the establishment of infection using the R62H/D77N strain. Cytotoxicity was decreased in the R62H/D77N strain, and the defect was restored by an in trans complementation. The intraperitoneal 50% lethal dose of the R62H/D77N strain increased by 26- and 238,000-fold in normal and iron-overloaded mice, respectively. The growth of the R62H/D77N strain in 50% HeLa cell lysate, 100% human ascitic fluid, and 50% human serum was significantly retarded compared to that of the isogenic wild-type strain. The R62H/D77N mutant also had a critical defect in the ability to survive and replicate even in iron-overloaded mice. These results demonstrate that pyrH is essential for the in vivo survival and growth of V. vulnificus and should be an attractive new target for the development of antibacterial drugs and replication-controllable live attenuated vaccines.

Cloning and characterization of the dxs gene, encoding 1-deoxy-d-xylulose 5-phosphate synthase from Agrobacterium tumefaciens, and its overexpression in Agrobacterium tumefaciens.

A newly isolated gene dxs11 from Agrobacterium tumefaciens (KCCM 10413), an organism with potential for the industrial production of ubiquinone-10 (UbiQ(10)), encoding a 1-deoxy-d-xylulose 5-phosphate synthase (Dxs), was cloned in Escherichia coli and its nucleotide sequence was determined. DNA sequence analysis revealed an open reading frame of 1920bp, capable of encoding a polypeptide of 640 amino acids residues with a calculated isoelectric point of pH 5.63 and a molecular mass of 68,054Da. The homodimeric enzyme was overexpressed in E. coli and purified as an active soluble form. The enzyme required thiamine diphosphate and a divalent metal ion, either Mg(2+) or Mn(2+), for enzymatic activity. The enzyme had an optimal pH and temperature of 8.0 and 37 degrees C, respectively, with a k(cat) of 26.8s(-1) and a k(cat)/K(m) of 0.67 and 1.17s(-1)M(-1) for pyruvate and d-glyceraldehyde 3-phosphate, respectively. A. tumefaciens Dxs showed a comparable catalytic efficiency to other Dxs proteins. The dxs11 gene was transformed into A. tumefaciens KCCM 10413, and the resulting recombinant, A. tumefaciens pGX11, showed higher UbiQ(10) production (502.4mg/l) and content (8.3mg/gDCW) than A. tumefaciens KCCM 10413, by 21.9 and 23.9%, respectively. This work describes Dxs from A. tumefaciens, an organism with the potential for industrial UbiQ(10) production, and the first metabolic engineering study with the non-mevalonate pathway enzyme in A. tumefaciens.

The CRH family coding for cell wall glycosylphosphatidylinositol proteins with a predicted transglycosidase domain affects cell wall organization and virulence of Candida albicans.

In Candida albicans UTR2 (CSF4), CRH11, and CRH12 are members of a gene family (the CRH family) that encode glycosylphosphatidylinositol-dependent cell wall proteins with putative transglycosidase activity. Deletion of genes of this family resulted in additive sensitivity to compounds interfering with normal cell wall formation (Congo red, calcofluor white, SDS, and high Ca(2+) concentrations), suggesting that these genes contribute to cell wall organization. A triple mutant lacking UTR2, CRH11, and CRH12 produced a defective cell wall, as inferred from increased sensitivity to cell wall-degrading enzymes, decreased ability of protoplasts to regenerate a new wall, constitutive activation of Mkc1p, the mitogen-activated protein kinase of the cell wall integrity pathway, and an increased chitin content of the cell wall. Importantly, this was accompanied by a decrease in alkali-insoluble 1,3-beta-glucan but not total glucan content, suggesting that formation of the linkage between 1,3-beta-glucan and chitin might be affected. In support of this idea, localization of a Utr2p-GFP fusion protein largely coincided with areas of chitin incorporation in C. albicans. As UTR2 and CRH11 expression is regulated by calcineurin, a serine/threonine protein phosphatase involved in tolerance to antifungal drugs, cell wall morphogenesis, and virulence, this points to a possible relationship between calcineurin and the CRH family. Deletion of UTR2, CRH11, and CRH12 resulted in only a partial overlap with calcineurin-dependent phenotypes, suggesting that calcineurin has additional targets. Interestingly, cells deleted for UTR2, CRH11, and CRH12 were, like a calcineurin mutant, avirulent in a mouse model of systemic infection but retained the capacity to colonize target organs (kidneys) as the wild type. In conclusion, this work establishes the role of UTR2, CRH11, and CRH12 in cell wall organization and integrity.

Assays of protein palmitoylation.

Protein palmitoylation plays an important role in the structure and function of a wide array of proteins. Unlike other lipid modifications, protein palmitoylation is highly dynamic and cycles of palmitoylation and depalmitoylation can regulate protein function and localization. The dynamic nature of palmitoylation is poorly resolved because of limitations in assay methods. Here, we discuss various methods that can be used to measure protein palmitoylation and identify sites of palmitoylation. We describe new methodology based on "fatty acyl exchange labeling" in which palmitate is removed via hydroxylamine-mediated cleavage of the palmitoyl-thioester bond and then exchanged with a sulfhydryl-specific labeling compound. The techniques are highly sensitive and allow for quantitative estimates of palmitoylation. Unlike other techniques used to assay posttranslational modifications, the techniques we have developed can label all sites of modification with a variety of probes, radiolabeled or non-radioactive, and can be used to assay the palmitoylation of proteins from tissue samples.

Proteomic identification of palmitoylated proteins.

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.

The analysis of cell division and cell wall synthesis genes reveals mutationally inactivated ftsQ and mraY in a protoplast-type L-form of Escherichia coli.

Cell division and cell wall synthesis are tightly linked cellular processes for bacterial growth. A protoplast-type L-form Escherichia coli, strain LW1655F+, indicated that bacteria can divide without assembling a cell wall. However, the molecular basis of its phenotype remained unknown. To establish a first phenotype-genotype correlation, we analyzed its dcw locus, and other genes involved in division of E. coli. The analysis revealed defective ftsQ and mraY genes, truncated by a nonsense and a frame-shift mutation, respectively. Missense mutations were determined in the ftsA and ftsW products yielding amino-acid replacements at conserved positions. FtsQ and MraY, obviously nonfunctional in the L-form, are essential for cell division and cell wall synthesis, respectively, in all bacteria with a peptidoglycan-based cell wall. LW1655F+ is able to survive their loss-of-functions. This points to compensatory mechanisms for cell division in the absence of murein sacculus formation. Hence, this L-form represents an interesting model to investigate the plasticity of cell division in E. coli, and to demonstrate how concepts fundamental for bacterial life can be bypassed.

Protein function microarrays based on self-immobilizing and self-labeling fusion proteins.

Protein microarrays are an attractive approach for the high-throughput analysis of protein function, but their impact on proteomics has been limited by the technical difficulties associated with their generation. Here we demonstrate that fusion proteins of O6-alkylguanine-DNA alkyltransferase (AGT) can be used for the simple and reliable generation of protein microarrays for the analysis of protein function. Important features of the approach are the selectivity of the covalent immobilization; this allows for direct immobilization of proteins out of cell extracts, and the option both to label and to immobilize AGT fusion proteins, which allows for direct screening for protein-protein interactions between different AGT fusion proteins. In addition to the identification of protein-protein interactions, AGT-based protein microarrays can be used for the characterization of small molecule-protein interactions or post-translational modifications. The potential of the approach was demonstrated by investigating the post-translational modification of acyl carrier protein (ACP) from E. coli by different phosphopantetheine transferases (PPTases), yielding insights into the role of selected ACP amino acids in the ACP-PPTase interaction.

Presence of free myxol and 4-hydroxymyxol and absence of myxol glycosides in Anabaena variabilis ATCC 29413, and proposal of a biosynthetic pathway of carotenoids.

We identified the molecular structures of all carotenoids in Anabaena variabilis ATCC 29413 (= IAM M-204). The major carotenoids were beta-carotene, echinenone and canthaxanthin. Myxol glycosides were absent, while free forms of myxol and 4-hydroxymyxol were present. The 4-hydroxyl group of the latter was a mixture of (4R) and (4S) configurations, which is a rare mixture in carotenoids. Thus, this strain was the first cyanobacterium found to have free myxol and not myxol glycosides, and seemed to lack the gene for or activity of glycosyl transferase. In another strain of A. variabilis IAM M-3 (= PCC 7118), we recently identified (3R,2'S)-myxol 2'-fucoside and (3S,2'S)-4-ketomyxol 2'-fucoside, and hence the strain ATCC 29413 might be useful for investigating the characteristics of myxol glycosides in cyanobacteria. Based on the identification of the carotenoids and the completion of the entire nucleotide sequence of the genome in A. variabilis ATCC 29413, we proposed a biosynthetic pathway of the carotenoids and the corresponding genes and enzymes. The homologous genes were searched by sequence homology only from the functionally confirmed genes.