Chain Length Determination Research Articles

Secretion genes as determinants of Bacillus anthracis chain length.

Bacillus anthracis grows in chains of rod-shaped cells, a trait that contributes to its escape from phagocytic clearance in host tissues. Using a genetic approach to search for determinants of B. anthracis chain length, we identified mutants with insertional lesions in secA2. All isolated secA2 mutants exhibited an exaggerated chain length, whereas the dimensions of individual cells were not changed. Complementation studies revealed that slaP (S-layer assembly protein), a gene immediately downstream of secA2 on the B. anthracis chromosome, is also a determinant of chain length. Both secA2 and slaP are required for the efficient secretion of Sap and EA1 (Eag), the two S-layer proteins of B. anthracis, but not for the secretion of S-layer-associated proteins or of other secreted products. S-layer assembly via secA2 and slaP contributes to the proper positioning of BslO, the S-layer-associated protein, and murein hydrolase, which cleaves septal peptidoglycan to separate chains of bacilli. SlaP was found to be both soluble in the bacterial cytoplasm and associated with the membrane. The purification of soluble SlaP from B. anthracis-cleared lysates did not reveal a specific ligand, and the membrane association of SlaP was not dependent on SecA2, Sap, or EA1. We propose that SecA2 and SlaP promote the efficient secretion of S-layer proteins by modifying the general secretory pathway of B. anthracis to transport large amounts of Sap and EA1.

Determination of the Critical Chain Length of Oligomers in Dispersion Polymerization.

The critical chain length (jcrit) in dispersion polymerization is systematically investigated utilizing low molecular weight poly(methyl methacrylate) oligomers synthesized by catalytic chain transfer polymerization. The solubility of these oligomers in methanol/water media of different compositions and at different temperatures has been visually determined. The results show that the solubility of the oligomers increases with increasing methanol fraction and increasing temperature. The constructed solubility map allows for an estimate of jcrit as a function of these important polymerization parameters. Furthermore, it is found that the value of jcrit changes with the concentration of the oligomers in the methanol/water medium, an important consideration for understanding the nucleation stage of a dispersion polymerization. The obtained results have been successfully correlated to earlier data reported on the dispersion polymerization of methyl methacrylate.

Structure-Guided Investigation of Lipopolysaccharide O-Antigen Chain Length Regulators Reveals Regions Critical for Modal Length Control

The O-antigen component of the lipopolysaccharide (LPS) represents a population of polysaccharide molecules with nonrandom (modal) chain length distribution. The number of the repeat O units in each individual O-antigen polymer depends on the Wzz chain length regulator, an inner membrane protein belonging to the polysaccharide copolymerase (PCP) family. Different Wzz proteins confer vastly different ranges of modal lengths (4 to >100 repeat units), despite having remarkably conserved structural folds. The molecular mechanism responsible for the selective preference for a certain number of O units is unknown. Guided by the three-dimensional structures of PCPs, we constructed a panel of chimeric molecules containing parts of two closely related Wzz proteins from Salmonella enterica and Shigella flexneri which confer different O-antigen chain length distributions. Analysis of the O-antigen length distribution imparted by each chimera revealed the region spanning amino acids 67 to 95 (region 67 to 95), region 200 to 255, and region 269 to 274 as primarily affecting the length distribution. We also showed that there is no synergy between these regions. In particular, region 269 to 274 also influenced chain length distribution mediated by two distantly related PCPs, WzzB and FepE. Furthermore, from the 3 regions uncovered in this study, region 269 to 274 appeared to be critical for the stability of the oligomeric form of Wzz, as determined by cross-linking experiments. Together, our data suggest that chain length determination depends on regions that likely contribute to stabilize a supramolecular complex.

Connected cavity structure enables prenyl elongation across the dimer interface in mutated geranylfarnesyl diphosphate synthase from Methanosarcina mazei

The mechanism for product chain-length determination of geranylfarnesyl diphosphate synthase from the methanogenic archaeon Methanosarcina mazei was investigated by constructing mutants based on structural information. Among the mutants, in which each of the bulky residues that constitute the bottom of the product-accommodating cavity was replaced with alanine, those having mutations on an α-helix existing at the subunit interface yielded longer products. In particular, replacement of isoleucine 112 on the α-helix greatly elongated the product chain-length, probably by connecting the reaction cavities of two subunits across the dimer interface.

Constraints on food chain length arising from regional metacommunity dynamics.

Classical ecological theory has proposed several determinants of food chain length, but the role of metacommunity dynamics has not yet been fully considered. By modelling patchy predator-prey metacommunities with extinction-colonization dynamics, we identify two distinct constraints on food chain length. First, finite colonization rates limit predator occupancy to a subset of prey-occupied sites. Second, intrinsic extinction rates accumulate along trophic chains. We show how both processes concur to decrease maximal and average food chain length in metacommunities. This decrease is mitigated if predators track their prey during colonization (habitat selection) and can be reinforced by top-down control of prey vital rates (especially extinction). Moreover, top-down control of colonization and habitat selection can interact to produce a counterintuitive positive relationship between perturbation rate and food chain length. Our results show how novel limits to food chain length emerge in spatially structured communities. We discuss the connections between these constraints and the ones commonly discussed, and suggest ways to test for metacommunity effects in food webs.

Enhanced Specificity of Mint Geranyl Pyrophosphate Synthase by Modifying the R-Loop Interactions

Isoprenoids, most of them synthesized by prenyltransferases (PTSs), are a class of important biologically active compounds with diverse functions. The mint geranyl pyrophosphate synthase (GPPS) is a heterotetramer composed of two LSU·SSU (large/small subunit) dimers. In addition to C 10-GPP, the enzyme also produces geranylgeranyl pyrophosphate (C 20-GGPP) in vitro, probably because of the conserved active-site structures between the LSU of mint GPPS and the homodimeric GGPP synthase from mustard. By contrast, the SSU lacks the conserved aspartate-rich motifs for catalysis. A major active-site cavity loop in the LSU and other trans-type PTSs is replaced by the regulatory R-loop in the SSU. Only C 10-GPP, but not C 20-GGPP, was produced when intersubunit interactions of the R-loop were disrupted by either deletion or multiple point mutations. The structure of the deletion mutant, determined in two different crystal forms, shows an intact (LSU·SSU) 2 heterotetramer, as previously observed in the wild-type enzyme. The active-site of LSU remains largely unaltered, except being slightly more open to the bulk solvent. The R-loop of SSU acts by regulating the product release from LSU, just as does its equivalent loop in a homodimeric PTS, which prevents the early reaction intermediates from escaping the active site of the other subunit. In this way, the product-retaining function of R-loop provides a more stringent control for chain-length determination, complementary to the well-established molecular ruler mechanism. We conclude that the R-loop may be used not only to conserve the GPPS activity but also to produce portions of C 20-GGPP in mint.

Loss of the WaaL O-Antigen Ligase Prevents Surface Activation of the Flagellar Gene Cascade in Proteus mirabilis

Proteus mirabilis is a Gram-negative bacterium that undergoes a physical and biochemical change from a vegetative swimmer cell (a typical Gram-negative rod) to an elongated swarmer cell when grown on a solid surface. In this study, we report that a transposon insertion in the waaL gene, encoding O-antigen ligase, blocked swarming motility on solid surfaces but had little effect on swimming motility in soft agar. The waaL mutant was unable to differentiate into a swarmer cell. Differentiation was also prevented by a mutation in wzz, encoding a chain length determinant for O antigen, but not by a mutation in wzyE, encoding an enzyme that polymerizes enterobacterial common antigen, a surface polysaccharide different from the lipid A::core. In wild-type P. mirabilis, increased expression of the flhDC operon occurs after growth on solid surfaces and is required for the high-level expression of flagellin that is characteristic of swarmer cells. However, in both the waaL and the wzz mutants, the flhDC operon was not activated during growth on agar. A loss-of-function mutation in the rcsB response regulator or overexpression of flhDC restored swarming to the waaL mutant, despite the absence of O antigen. Therefore, although O antigen may serve a role in swarming by promoting wettability, the loss of O antigen blocks a regulatory pathway that links surface contact with the upregulation of flhDC expression.

Substrate Specificities of <i>E</i>-and <i>Z</i>-Farnesyl Diphosphate Synthases with Artificial Substrates

Graduate School of Science and Technology, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori 036-8561, Japan *Department of Material and Biological Chemistry, Faculty of Science, Yamagata University, Kojirakawa-machi, Yamagata 990-8560, Japan **Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8577, Japan Fax: 81-172-39-3947, e-mail: nagaki@cc.hirosaki-u.ac.jp Here, to assess substrate specificities of E-and Z-farnesyl diphosphate synthases from Bacillus stearothermophilus and Thermobifida fusca, we examined the reactivities of isopentenyl diphosphate homologs having an alkyl group at the 3-position. E- and Z-FPP synthase reactions of geranyl diphosphate (GPP) with 3-ethylisopentenyl diphosphate produced E- and Z-3-ethylfarnesyl diphosphates (yield: 70.8%, and 11.3%, respectively). The E-FPP synthase reaction of GPP with 3-propylisopentenyl diphosphate produced E-3-propylfarnesyl diphosphate (yield: 40.1%); however, when reacting GPP with 3-propylisopentenyl diphosphate by Z-FPP synthase reaction, only trace product was acquired. Further, neither E- nor Z-FPP synthase reaction of GPP with 3-butylisopentanyl diphosphate produced any product. Key words: E- and Z-farnesyl diphosphate synthases, substrate specificity, homologs of isopentenyl diphosphate, Bacillus stearothermophilus, Thermobifida fusca 1. INTRODUCTION Prenyltransferases can be roughly divided into Z- and E-prenyl chain elongating enzymes[1-5]. E-farnesyl diphosphate (FPP) synthase, a short prenyl chain elongating enzyme, has long been the subject of intense research. The E-FPP synthase has been solved to quite detailed portions, such as a chain length determination mechanism [6-8]. However, after Crick et al. successfully cloned Z-FPP synthase from Mycobacterium tubercule in 2000, a great number of studies began focusing on Z-prenyltransferase[9-11]. Subsequent to Crick et al.’s findings, Koyama et al. recently successfully cloned Z-FPP synthase from several other bacterial species, including Thermobifida fusca and Corynebacterium glutamicum [12-14]. C. Sallaud et al. identified the genes encoding Z-FPP synthase in the wild tomato, Solanum habrochaites, and reported that the recombinant enzymatic reaction between dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP) produced all-Z-FPP [15]. E-FPP synthase [EC 2.5.1.10] catalyzes the head-to-tail condensation of IPP with DMAPP (or geranyl diphosphate [GPP]) as an allylic substrate, producing FPP as the final product (Scheme 1). Several previous studies have investigated the substrate specificities of FPP synthase with both allylic and homoallylic substrate homologs [16-20]. Scheme 1. FPP Synthase Reaction of DMAPP withIPP Our team also recently reported on the reactivities of several allylic substrate homologs which have a hydrophilic group at the ω-position, using B. stearothermophilus and porcine liver FPP synthases [21-23]. Previously, to examine the substrate specificity of FPP synthase derived from B. stearothermophilus, we reported on the reaction of GPP with IPP homologs which have an alkyl group at the 3-position[24]. Here, we compared E- and Z-FPP synthases by examining several new, similar reactions. We then assessed the substrate specificities of the synthases by examining the reactivity of artificial substrate homologs.

Structural features conferring dual Geranyl/Farnesyl diphosphate synthase activity to an aphid prenyltransferase

In addition to providing lipid chains for protein prenylation, short-chain isoprenyl diphosphate synthases (scIPPSs) play a pivotal role in the biosynthesis of numerous mevalonate pathway end-products, including insect juvenile hormone and terpenoid pheromones. For this reason, they are being considered as targets for pesticide development. Recently, we characterized an aphid scIPPS displaying dual geranyl diphosphate (GPP; C 10)/farnesyl diphosphate (FPP; C 15) synthase activity in vitro. To identify the mechanism(s) responsible for this dual activity, we assessed the product selectivity of aphid scIPPSs bearing mutations at Gln107 and/or Leu110, the fourth and first residue upstream from the “first aspartate-rich motif” (FARM), respectively. All but one resulted in significant changes in product chain-length selectivity, effectively increasing the production of either GPP (Q107E, L110W) or FPP (Q107F, Q107F–L110A); the other mutation (L110A) abolished activity. Although some of these effects could be attributed to changes in steric hindrance within the catalytic cavity, molecular dynamics simulations identified other contributing factors, including residue-ligand Van der Waals interactions and the formation of hydrogen bonds or salt bridges between Gln107 and other residues across the catalytic cavity, which constitutes a novel product chain-length determination mechanism for scIPPSs. Thus the aphid enzyme apparently evolved to maintain the capacity to produce both GPP and FPP through a balance between these mechanisms.

Mechanism of Chain Length Determination in Biosynthesis of Milk Fatty Acids

Fatty acid synthetases isolated from all mammalian tissues synthesize pre-dominantly palmitic acid. However, in vivo the mammary gland fatty acid synthetases of some species are responsible for the synthesis of medium chain fatty acids. The objective of this presentation is to outline the mechanism which regulates the product specificity of fatty acid synthetases in general and to illustrate how this control is modified in the mammary gland. Fatty acid synthetases isolated from mammalian tissues are composed of two similar, probably identical, polypeptides, each carrying as many as seven enzyme components. Thioesterase I, the component which functions to terminate growth of acyl chains of the multienzyme, is located at one terminus of each polyfunctional polypeptide and can be detached by limited proteolysis with trypsin. By studying separately the kinetics of chain elongation by the core of the trypsinized complex and of chain termination by the chain thioesterase I component, it has been possible to establish that the specificities of the elongation and termination reactions account for the synthesis of predominantly the carbon-16 fatty acid by purified fatty acid synthetases. Mammary glands for some species contain an additional enzyme, thioesterase II, which can modify the product specificity of fatty acid sunthetase by hydrolyzing medium chain acyl moieties from thioester linkage to the 4'phosphopantetheine of the multienzyme. At all stages of development of rat mammary gland, the amount of theoesterase II present correlates with the proportion of medium chain fatty acids synthesized by the gland. This mammary glad-specific thioesterase appears responsible for the ability of this tissue to synthesize medium chain fatty acids characteristic of milk fat.

Introduction: Milk Lipid Synthesis: Chain Length Determination and Secretory Differentiation

In 1967 Stuart Smith and R. Watts, working with RayDils at the National Institute for Research in Dairying atReading, found that there was very little difference inthe fatty acid composition of the adipose tissue ofdifferent species, while in contrast milk fat had anaverage fatty acid chain length of 11.7 in rabbits, 14.2 inrats,15.3inmice,and17.2inguineapigs[1]. Smithand Dils had previously shown that fatty acids with achain length from 6 to 14 carbons could be synthesized bymammary tissue from lactating rabbits, making it clearthat de novo fatty acid synthesis in the mammary glanddiffered fundamentally from that in other organs [2].Evidence for an independent tissue specific factor in themammary gland that terminated chain elongation earlywas obtained in several laboratories [3]. By 1969 Smithhad moved to the Children’s Hospital Medical Center ofNorthCarolinatoworkwithSandyAbraham;bothheandRay Dils continued to work on this factor, which becameknown as thioesterase II. It was a difficult job, finallysuccessful in 1978 [4]. In the paper reproduced here, thepathway to purification of this factor as well as thegeneral mechanisms of fatty acid synthesis are described[5].Smith describes the circumstances under which hegave the talk at the 74th Annual Meeting of theAmerican Dairy Science in 1979 that resulted in thispublication: “That meeting, held on the campus of UtahStateUniversityinLogan,includedasymposiumonMilk Synthesis. I recall that Nick Kuhn from theUniversity of Birmingham, England, (my alma mater),also gave a talk on lactose synthesis. I don't remembermany details of the meeting except that I arrived at sunseton a small prop plane from Salt Lake City to find that thetiny airport was completely deserted, not a taxi in sight. Iwas rescued by the pilot of our plane who drove me in hiscar directly to my hotel in town. Now that's service youwill not find anywhere these days!”He goes on: “During the five year period leading upto the talk it was established that the animal fatty acidsynthase was a ‘multifunctional polypeptide’ in whichall of the enzymes of that biosynthetic pathway wereintegrated into a single polypeptide. Little did we knowthat it would take another 30 years before the detailedstructure of the complex would be established [6, 7].Nevertheless, at the time of the meeting in Logan, we hadcome to appreciate that all tissues use the same enzymesystem for de novo fatty acid biosynthesis (the fatty acidsynthase), regardless of the products made. In the case ofthe lactating mammary gland, the ability to makemedium-chain saturated fatty acids was dependent onthe presence of a tissue-specific thioesterase (termed‘thioesterase II’) that, remarkably, was able to interruptthe chain elongation process by accessing and hydrolyz-ing the thioester bond linking these intermediates to theprotein. We now know that the resident thioesterase(termed ‘thioesterase I’), which normally terminates chainelongation at 16 carbon atoms, is secured to its neighbor-ing domain by a relatively long flexible tether, whichpresumably allows it to be displaced by the externalthiosterase II thus allowing access to intermediate chainlength intermediates. Nevertheless, the details of how thisis achieved are still to be worked out.”One of the next big questions is how the activity ofthis enzyme is regulated. There is increasing evidence at

Reaction Kinetics, Catalytic Mechanisms, Conformational Changes, and Inhibitor Design for Prenyltransferases

Isoprenoids comprise a family of more than 55000 natural products with great structural variety derived from five-carbon isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP). Allylic diphosphates such as farnesyl diphosphate (FPP) synthesized from DMAPP and IPP serve as outlet points for a great variety of products. A group of prenyltransferases catalyzing chain elongation of FPP to designated lengths by consecutive condensation reactions with specific numbers of IPP are classified as cis and trans types according to the stereochemistry of the double bonds formed by IPP condensation. The complete kinetics of the multistep IPP condensation reactions by both types of enzymes has been determined using steady-state and pre-steady-state approaches. Because their crystal structures were determined in conjunction with biochemical studies, a more thorough understanding of their catalytic mechanisms, protein conformational changes, and product chain-length determination mechanisms has been gained recently. Since these prenyltransferases play important roles, potent inhibitors have been identified and their cocrystal structures have been determined for drug development. In this review, the current knowledge of these prenyltransferases that synthesize prenyl oligomers or polymers is summarized.

Characterization of the Lipid Linkage Region and Chain Length of the Cellubiuronic Acid Capsule of Streptococcus pneumoniae

The processive reaction mechanisms of beta-glycosyl-polymerases are poorly understood. The cellubiuronan synthase of Streptococcus pneumoniae catalyzes the synthesis of the type 3 capsular polysaccharide through the alternate additions of beta-1,3-Glc and beta-1,4-GlcUA. The processive multistep reaction involves the sequential binding of two nucleotide sugar donors in coordination with the extension of a polysaccharide chain associated with the carbohydrate acceptor recognition site. Degradation analysis using cellubiuronan-specific depolymerase demonstrated that the oligosaccharide-lipid and polysaccharide-lipid products synthesized in vitro with recombinant cellubiuronan synthase had a similar oligosaccharyl-lipid at their reducing termini, providing definitive evidence for a precursor-product relationship and also confirming that growth occurred at the nonreducing end following initiation on phosphatidylglycerol. The presence of a lipid marker at the reducing end allowed the quantitative determination of cellubiuronic acid polysaccharide chain lengths. As the UDP-GlcUA concentration was increased from 1 to 11.5 mum, the level of synthase in the transitory processive state decreased, with the predominant oligosaccharide-lipid product containing 3 uronic acid residues, whereas the proportion of synthase in the fully processive state increased and the polysaccharide chain length increased from 320 to 6700 monosaccharide units. In conjunction with other kinetic data, these results suggest that the formation of a complex between a tetrauronosyl oligomer and the carbohydrate acceptor recognition site plays a central role in coordinating the repetitive interaction of the synthase with the nucleotide sugar donors and modulating the chain length of cellubiuronan polysaccharide.

Biochemical and Structural Analysis of Bacterial O-antigen Chain Length Regulator Proteins Reveals a Conserved Quaternary Structure

Lipopolysaccharide (LPS) is a major component of the Gram-negative outer membrane and is an important virulence determinant. The O-antigen polysaccharide of the LPS molecule provides protection from host defenses, and the length of O-antigen chains plays a pivotal role. In the Wzy-dependent O-antigen biosynthesis pathway, the integral inner membrane protein Wzz determines the O-antigen chain length. How these proteins function is currently unknown, but the hypothesis includes activities such as a "molecular ruler" or a "molecular stopwatch," and other possibilities may exist. Wzz homologs are membrane proteins with two transmembrane helices that flank a large periplasmic domain. Recent x-ray crystallographic studies of the periplasmic portions of Wzz proteins found multiple oligomeric forms, with quaternary structures favoring the "molecular ruler" interpretation. Here, we have studied full-length Wzz proteins with the transmembrane portions embedded in lipid membranes. Using electron microscopy and image analysis we find a unique hexameric state rather than differing oligomeric forms. The data suggest that in vivo Wzz proteins determine O-antigen chain length via subtle structure-function relationships at the level of primary, secondary, or tertiary structure within the context of a hexameric complex.

Effect of mutagenesis at the region upstream from the G(Q/E) motif of three types of geranylgeranyl diphosphate synthase on product chain-length

(All-E) geranylgeranyl diphosphate synthases have been classified into three types based on the characteristic sequences around the first aspartate rich motif, which is highly conserved among the enzymes. In type I geranylgeranyl diphosphate synthases, which consist of archaeal enzymes, a bulky amino acid residue at the 5th position upstream from the motif plays a main role in the product determination, by blocking further elongation of prenyl chain as the bottom of the reaction cavity. On the other hand, type III geranylgeranyl diphosphate synthases, which consist of the enzymes from eukaryotes except for plants, use a bulky amino acid residue at the 2nd position upstream from the conserved G(Q/E) motif for product chain-length determination. Thus we introduced mutations into the region upstream from the G(Q/E) motif of geranylgeranyl diphosphate synthases of the three different types to confirm the importance of the region for the product chain-length determination. The results of the mutational analyses indicated that not only the 2nd but also the 3rd position upstream from the G(Q/E) motif is involved in the product chain-length determination mechanism in types I and III geranylgeranyl diphosphate synthases, while the amino acid substitution in this region did not affect the chain-length of the products of type II geranylgeranyl diphosphate synthase, which consist of the enzymes from bacteria and plants. The region upstream from the G(Q/E) motif possibly contributes to the product determination in the wide range of geranylgeranyl diphosphate synthases, as well as that around the first aspartate rich motif.

Targeting a Uniquely Nonspecific Prenyl Synthase with Bisphosphonates to Combat Cryptosporidiosis

Cryptosporidiosis is a neglected disease without a wholly effective drug. We present a study demonstrating nitrogen-containing bisphosphonates (N-BPs) to be capable of inhibiting Cryptosporidium parvum at low micromolar concentrations in infected MDCK cells. Predictably, the mechanism of action is based on inhibition of biosynthesis of isoprenoids but the target enzyme is unexpectedly a distinctive C. parvum enzyme dubbed nonspecific polyprenyl pyrophosphate synthase (CpNPPPS). This enzyme produces various isoprenoid products larger than FPP and is inhibited by N-BPs at subnanomolar concentrations. It is part of an isoprenoid pathway in Cryptosporidium distinctly different from other organisms. The proposed mechanism of action is corroborated by crystal structures of the enzyme with risedronate and zoledronate bound showing how this enzyme's unique chain length determinant region enables it to accommodate larger substrates and products. These results, combined with existing data on their clinical use, demonstrate that N-BPs are very promising anticryptosporidial drug candidates.

Product chain-length determination mechanism of Z, E-farnesyl diphosphate synthase

cis-Prenyltransferases catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyl diphosphates, producing Z, E-mixed prenyl diphosphate. The Mycobacterium tuberculosis Z, E-farnesyl diphosphate synthase Rv1086 catalyzes the condensation of one molecule of IPP with geranyl diphosphate to yield Z, E-farnesyl diphosphate and is classified as a short-chain cis-prenyltransferase. To elucidate the chain-length determination mechanism of the short-chain cis-prenyltransferase, we introduced some substitutive mutations at the characteristic amino acid residues of Rv1086. Among the mutants constructed, L84A showed a dramatic change of catalytic function to synthesize longer prenyl chain products than that of wild type, indicating that Leu84 of Rv1086 plays an important role in product chain-length determination. Mutagenesis at the corresponding residue of a medium-chain cis-prenyltransferase, Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase also resulted in the production of different prenyl chain length from the intrinsic product, suggesting that this position also plays an important role in product chain-length determination for medium-chain cis-prenyltransferases.

Cloning and functional analysis of novel short-chain cis-prenyltransferases

cis-Prenyltransferase catalyzes the synthesis of Z,E-mixed prenyl diphosphates by sequential condensation of isopentenyl diphosphate with allylic diphosphate. cis-Prenyltransferases can be classified into three subgroups: short-, medium-, and long-chain cis-prenyltransferase, according to their product chain lengths. cis-Farnesyl diphosphate synthase from Mycobacterium tuberculosis has been the only example as short-chain cis-prenyltransferase so far characterized. In this study, we cloned the novel short-chain cis-prenyltransferases from three different bacteria, and characterized their enzymatic activities to compare and elucidate a common feature of the short-chain cis-prenyltransferases. Furthermore, we identified a specific isoleucine that is conserved in short-chain cis-prenyltransferases and located in close proximity of the ω-end of the geranyl diphosphate. Several site-directed mutants with respect to the isoleucine residue synthesized longer prenyl chain products and showed broader allylic substrate specificity. These results suggested that the isoleucine plays an important role in the substrate specificity and chain length determination mechanism of cis-prenyltransferase.

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase requires subunit interaction

The product chain length determination mechanism of type II geranylgeranyl diphosphate synthase from the bacterium, Pantoea ananatis, was studied. In most types of short-chain (all-E) prenyl diphosphate synthases, bulky amino acids at the fourth and/or fifth positions upstream from the first aspartate-rich motif play a primary role in the product determination mechanism. However, type II geranylgeranyl diphosphate synthase lacks such bulky amino acids at these positions. The second position upstream from the G(Q/E) motif has recently been shown to participate in the mechanism of chain length determination in type III geranylgeranyl diphosphate synthase. Amino acid substitutions adjacent to the residues upstream from the first aspartate-rich motif and from the G(Q/E) motif did not affect the chain length of the final product. Two amino acid insertion in the first aspartate-rich motif, which is typically found in bacterial enzymes, is thought to be involved in the product determination mechanism. However, deletion mutation of the insertion had no effect on product chain length. Thus, based on the structures of homologous enzymes, a new line of mutants was constructed in which bulky amino acids in the alpha-helix located at the expected subunit interface were replaced with alanine. Two mutants gave products with longer chain lengths, suggesting that type II geranylgeranyl diphosphate synthase utilizes an unexpected mechanism of chain length determination, which requires subunit interaction in the homooligomeric enzyme. This possibility is strongly supported by the recently determined crystal structure of plant type II geranylgeranyl diphosphate synthase.

Structural Analysis of Farnesyl Pyrophosphate Synthase from Parasitic Protozoa, a Potential Chemotherapeutic Target

Synthesis of farnesyl pyrophosphate (FPP), a key intermediate of the isoprenoid biosynthesis pathway, is catalyzed by FPP synthase (FPPS). Antiprotozoal properties of bisphosphonates, which target FPPS, have generated interest in FPPS as a potential antiprotozoal drug target. The genes encoding FPPS from parasitic protozoa were assessed to analyze structural and functional features of the enzyme. Comparisons of the FPPS from the parasitic protozoa and search for conserved motifs revealed that FPPS from both apicomplexan and trypanosomatid parasites show characteristic conserved regions for example first aspartate rich motif (FARM) contained within II conserved domain and the second aspartate rich motif (SARM) contained within VI conserved domain. Phylogenetic analysis of FPPS generated a tree with three distinct clusters. Overall topology of the phylogenic tree constructed with small subunit ribosomal RNA sequences was almost similar to that constructed with FPPS sequences. Comparative homology modeling and structural comparisons of FPPS from the parasitic protozoa provided significant insights into common and distinct characteristics of the enzyme. The critical interacting residues of the isopentenyl pyrophosphate binding site are conserved across the enzymes from the family except for malarial FPPS where the C-terminal residues from the BXB motif of helix J were missing. Variations noticed in aromatic residue pairs at the fourth and fifth position upstream of the FARM, which play important role in determination of chain length of the polyprenyl products, may produce functional differences among protozoan FPPSs. The structural comparison of protozoan FPPS may be useful in designing common or selective FPPS inhibitors as potential broad spectrum or selective antiprotozoal agents.