Making iron-sulfur cluster: structure, regulation and evolution of the bacterial ISC system.

Iron sulfur (Fe-S) clusters rank among the most ancient and conserved prosthetic groups. Fe-S clusters containing proteins are present in most, if not all, organisms. Fe-S clusters containing proteins are involved in a wide range of cellular processes, from gene regulation to central metabolism, via gene expression, RNA modification or bioenergetics. Fe-S clusters are built by biogenesis machineries conserved throughout both prokaryotes and eukaryotes. We focus mostly on bacterial ISC machinery, but not exclusively, as we refer to eukaryotic ISC system when it brings significant complementary information. Besides covering the structural and regulatory aspects of Fe-S biogenesis, this review aims to highlight Fe-S biogenesis facets remaining matters of discussion, such as the role of frataxin, or the link between fatty acid metabolism and Fe-S homeostasis. Last, we discuss recent advances on strategies used by different species to make and use Fe-S clusters in changing redox environmental conditions.

Uncoordinated (UNC)119: coordinating the trafficking of myristoylated proteins.

The mechanism by which myristoylated proteins are targeted to specific subcellular membrane compartments is poorly understood. Two novel acyl-binding proteins, UNC119A and UNC119B, have been shown recently to function as chaperones/co-factors in the transport of myristoylated G protein α-subunits and src-type tyrosine kinases. UNC119 polypeptides feature an immunoglobulin-like ß-sandwich fold that forms a hydrophobic pocket capable of binding lauroyl (C12) and myristoyl (C14) side chains. UNC119A in rod photoreceptors facilitates the transfer of transducin α subunits (Tα) from inner segment to outer segment membranes by forming an intermediate diffusible UNC119-Tα complex. Similar complexes are formed in other sensory neurons, as the G proteins ODR-3 and GPA-13 in Caenorhabditis elegans unc-119 mutants traffic inappropriately. UNC119B knockdown in IMCD3 cells prevents trafficking ofmyristoylated nephrocystin-3 (NPHP3), a protein associated with nephronophthisis, to cilia. Further, UNC119A was shown to transport myristoylated src-type tyrosine kinases to cell membranes and to affect T-cell receptor (TCR) and interleukin-5 receptor (IL-5R) activities. These interactions establish UNC119 polypeptides as novel lipid-binding chaperones with specificity for a diverse subset of myristoylated proteins.

Current understanding of fatty acid biosynthesis and the acyl carrier protein.

FA (fatty acid) synthesis represents a central, conserved process by which acyl chains are produced for utilization in a number of end-products such as biological membranes. Central to FA synthesis, the ACP (acyl carrier protein) represents the cofactor protein that covalently binds all fatty acyl intermediates via a phosphopantetheine linker during the synthesis process. FASs (FA synthases) can be divided into two classes, type I and II, which are primarily present in eukaryotes and bacteria/plants respectively. They are characterized by being composed of either large multifunctional polypeptides in the case of type I or consisting of discretely expressed mono-functional proteins in the type II system. Owing to this difference in architecture, the FAS system has been thought to be a good target for the discovery of novel antibacterial agents, as exemplified by the antituberculosis drug isoniazid. There have been considerable advances in this field in recent years, including the first high-resolution structural insights into the type I mega-synthases and their dynamic behaviour. Furthermore, the structural and dynamic properties of an increasing number of acyl-ACPs have been described, leading to an improved comprehension of this central carrier protein. In the present review we discuss the state of the understanding of FA synthesis with a focus on ACP. In particular, developments made over the past few years are highlighted.

Functional characterization of the acyl-[acyl carrier protein] ligase in the Cryptosporidium parvum giant polyketide synthase.

The apicomplexan Cryptosporidium parvum possesses a unique 1500-kDa polyketide synthase (CpPKS1) comprised of 29 enzymes for synthesising a yet undetermined polyketide. This study focuses on the biochemical characterization of the 845-amino acid loading unit containing acyl-[ACP] ligase (AL) and acyl carrier protein (ACP). The CpPKS1-AL domain has a substrate preference for long chain fatty acids, particularly for the C20:0 arachidic acid. When using [3H]palmitic acid and CoA as co-substrates, the AL domain displayed allosteric kinetics towards palmitic acid (Hill coefficient, h=1.46, K50=0.751 microM, Vmax=2.236 micromol mg(-1) min(-1)) and CoA (h=0.704, K50=5.627 microM, Vmax=0.557 micromol mg(-1) min(-1)), and biphasic kinetics towards adenosine 5'-triphosphate (Km1=3.149 microM, Vmax1=373.3 nmol mg(-1) min(-1), Km2=121.0 microM, and Vmax2=563.7 nmol mg(-1) min(-1)). The AL domain is Mg2+-dependent and its activity could be inhibited by triacsin C (IC50=6.64 microM). Furthermore, the ACP domain within the loading unit could be activated by the C. parvum surfactin production element-type phosphopantetheinyl transferase. After attachment of the fatty acid substrate to the AL domain for conversion into the fatty-acyl intermediate, the AL domain is able to transfer palmitic acid to the activated holo-ACP in vitro. These observations ultimately validate the function of the CpPKS1-AL-ACP unit, and make it possible to further dissect the function of this megasynthase using recombinant proteins in a stepwise procedure.

Acyl carrier protein is a cellular target for the antibacterial action of the pantothenamide class of pantothenate antimetabolites.

Pantothenate is the precursor of the essential cofactor coenzyme A (CoA). Pantothenate kinase (CoaA) catalyzes the first and regulatory step in the CoA biosynthetic pathway. The pantothenate analogs N-pentylpantothenamide and N-heptylpantothenamide possess antibiotic activity against Escherichia coli. Both compounds are substrates for E. coli CoaA and competitively inhibit the phosphorylation of pantothenate. The phosphorylated pantothenamides are further converted to CoA analogs, which were previously predicted to act as inhibitors of CoA-dependent enzymes. Here we show that the mechanism for the toxicity of the pantothenamides is due to the inhibition of fatty acid biosynthesis through the formation and accumulation of the inactive acyl carrier protein (ACP), which was easily observed as a faster migrating protein using conformationally sensitive gel electrophoresis. E. coli treated with the pantothenamides lost the ability to incorporate [1-(14)C]acetate to its membrane lipids, indicative of the inhibition of fatty acid synthesis. Cellular CoA was maintained at the level sufficient for bacterial protein synthesis. Electrospray ionization time-of-flight mass spectrometry confirmed that the inactive ACP was the product of the transfer of the inactive phosphopantothenamide moiety of the CoA analog to apo-ACP, forming the ACP analog that lacks the sulfhydryl group for the attachment of acyl chains for fatty acid synthesis. Inactive ACP accumulated in pantothenamide-treated cells because of the active hydrolysis of regular ACP and the slow turnover of the inactive prosthetic group. Thus, the pantothenamides are pro-antibiotics that inhibit fatty acid synthesis and bacterial growth because of the covalent modification of ACP.

The pro1(+) gene from Sordaria macrospora encodes a C6 zinc finger transcription factor required for fruiting body development.

During sexual morphogenesis, the filamentous ascomycete Sordaria macrospora differentiates into multicellular fruiting bodies called perithecia. Previously it has been shown that this developmental process is under polygenic control. To further understand the molecular mechanisms involved in fruiting body formation, we generated the protoperithecia forming mutant pro1, in which the normal development of protoperithecia into perithecia has been disrupted. We succeeded in isolating a cosmid clone from an indexed cosmid library, which was able to complement the pro1(-) mutation. Deletion analysis, followed by DNA sequencing, subsequently demonstrated that fertility was restored to the pro1 mutant by an open reading frame encoding a 689-amino-acid polypeptide, which we named PRO1. A region from this polypeptide shares significant homology with the DNA-binding domains found in fungal C6 zinc finger transcription factors, such as the GAL4 protein from yeast. However, other typical regions of C6 zinc finger proteins, such as dimerization elements, are absent in PRO1. The involvement of the pro1(+) gene in fruiting body development was further confirmed by trying to complement the mutant phenotype with in vitro mutagenized and truncated versions of the pro1 open reading frame. Southern hybridization experiments also indicated that pro1(+) homologues are present in other sexually propagating filamentous ascomycetes.