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1.
In plants, peroxisomes are the organelles involved in various metabolic processes and physiological functions including β-oxidation, mobilization of seed storage lipids, photorespiration, and hormone biosynthesis. We have recently shown that, in fungi and plants, peroxisomes play a vital role in biosynthesis of biotin, an essential cofactor required for various carboxylation and decarboxylation reactions. In fungi, the mutants defective in peroxisomal protein import exhibit biotin auxotrophy. The fungal BioF protein, a 7-keto-8-aminopelargonic acid (KAPA) synthase catalyzing the conversion of pimeloyl-CoA to KAPA in biotin biosynthesis, contains the peroxisomal targeting sequence 1 (PTS1), and its peroxisomal targeting is required for biotin biosynthesis. In plants, biotin biosynthesis is essential for embryo development. We have shown that the peroxisomal targeting sequences of the BioF proteins are conserved throughout the plant kingdom, and the Arabidopsis thaliana BioF protein is indeed localized in peroxisomes. Our findings suggest that peroxisomal localization of the BioF protein is evolutionarily conserved among eukaryotes, and required for biotin biosynthesis and plant growth and development.  相似文献   

2.
Trl1 is an essential 827 amino acid enzyme that executes the end-healing and end-sealing steps of tRNA splicing in Saccharomyces cerevisiae. Trl1 consists of two domains—an N-terminal ligase component and a C-terminal 5′-kinase/2′,3′-cyclic phosphodiesterase (CPD) component—that can function in tRNA splicing in vivo when expressed as separate polypeptides. To understand the structural requirements for the kinase-CPD domain, we performed an alanine scan of 30 amino acids that are conserved in Trl1 homologs from other fungi. We thereby identified four residues (Arg463, His515, Thr675 and Glu741) as essential for activity in vivo. Structure–function relationships at these positions, and at four essential or conditionally essential residues defined previously (Asp425, Arg511, His673 and His777), were clarified by introducing conservative substitutions. Biochemical analysis showed that lethal mutations of Asp425, Arg463, Arg511 and His515 in the kinase module abolished polynucleotide kinase activity in vitro. We report that a recently cloned 1104 amino acid Arabidopsis RNA ligase functions in lieu of yeast Trl1 in vivo and identify essential side chains in the ligase, kinase and CPD modules of the plant enzyme. The plant ligase, like yeast Trl1 but unlike T4 RNA ligase 1, requires a 2′-PO4 end for tRNA splicing in vivo.  相似文献   

3.

Background  

Biotin is an essential enzyme cofactor that acts as a CO2 carrier in carboxylation and decarboxylation reactions. The E. coli genome encodes a biosynthetic pathway that produces biotin from pimeloyl-CoA in four enzymatic steps. The final step, insertion of sulfur into desthiobiotin to form biotin, is catalyzed by the biotin synthase, BioB. A dedicated biotin ligase (BirA) catalyzes the covalent attachment of biotin to biotin-dependent enzymes. Isotopic labeling has been a valuable tool for probing the details of the biosynthetic process and assaying the activity of biotin-dependent enzymes, however there is currently no established method for 35S labeling of biotin.  相似文献   

4.

Background

In the 1950s, Reed and coworkers discovered an enzyme activity in Streptococcus faecalis (Enterococcus faecalis) extracts that inactivated the Escherichia. coli and E. faecalis pyruvate dehydrogenase complexes through cleavage of the lipoamide bond. The enzyme that caused this lipoamidase activity remained unidentified until Jiang and Cronan discovered the gene encoding lipoamidase (Lpa) through the screening of an expression library. Subsequent cloning and characterization of the recombinant enzyme revealed that lipoamidase is an 80 kDa protein composed of an amidase domain containing a classic Ser-Ser-Lys catalytic triad and a carboxy-terminal domain of unknown function. Here, we show that the amidase domain can be used as an in vivo probe which specifically inactivates lipoylated enzymes.

Methodology/Principal Findings

We evaluated whether Lpa could function as an inducible probe of α-ketoacid dehydrogenase inactivation using E. coli as a model system. Lpa expression resulted in cleavage of lipoic acid from the three lipoylated proteins expressed in E. coli, but did not result in cleavage of biotin from the sole biotinylated protein, the biotin carboxyl carrier protein. When expressed in lipoylation deficient E. coli, Lpa is not toxic, indicating that Lpa does not interfere with any other critical metabolic pathways. When truncated to the amidase domain, Lpa retained lipoamidase activity without acquiring biotinidase activity, indicating that the carboxy-terminal domain is not essential for substrate recognition or function. Substitution of any of the three catalytic triad amino acids with alanine produced inactive Lpa proteins.

Conclusions/Significance

The enzyme lipoamidase is active against a broad range of lipoylated proteins in vivo, but does not affect the growth of lipoylation deficient E. coli. Lpa can be truncated to 60% of its original size with only a partial loss of activity, resulting in a smaller probe that can be used to study the effects of α-ketoacid dehydrogenase inactivation in vivo.  相似文献   

5.
Fatty acid biosynthesis is essential for bacterial survival. Components of this biosynthetic pathway have been identified as attractive targets for the development of new antibacterial agents. FabH, β-ketoacyl-acyl carrier protein (ACP) synthase III, is a particularly attractive target, since it is central to the initiation of fatty acid biosynthesis and is highly conserved among Gram positive and negative bacteria. Three series of Schiff bases containing thiazole template were synthesized and developed as potent inhibitors of FabH. This inhibitor class demonstrates strong antibacterial activity. Escherichia coli FabH inhibitory assay and docking simulation indicated that the compounds 11 and 18 were potent inhibitors of E. coli FabH.  相似文献   

6.
We recently identified a gene (FTN_0818) required for Francisella virulence that seemed likely involved in biotin metabolism. However, the molecular function of this virulence determinant was unclear. Here we show that this protein named BioJ is the enzyme of the biotin biosynthesis pathway that determines the chain length of the biotin valeryl side‐chain. Expression of bioJ allows growth of an Escherichia coli bioH strain on biotin‐free medium, indicating functional equivalence of BioJ to the paradigm pimeloyl‐ACP methyl ester carboxyl‐esterase, BioH. BioJ was purified to homogeneity, shown to be monomeric and capable of hydrolysis of its physiological substrate methyl pimeloyl‐ACP to pimeloyl‐ACP, the precursor required to begin formation of the fused heterocyclic rings of biotin. Phylogenetic analyses confirmed that distinct from BioH, BioJ represents a novel subclade of the α/β‐hydrolase family. Structure‐guided mapping combined with site‐directed mutagenesis revealed that the BioJ catalytic triad consists of Ser151, Asp248 and His278, all of which are essential for activity and virulence. The biotin synthesis pathway was reconstituted reaction in vitro and the physiological role of BioJ directly assayed. To the best of our knowledge, these data represent further evidence linking biotin synthesis to bacterial virulence.  相似文献   

7.
Biotin synthesis in Escherichia coli requires the functions of the bioH and bioC genes to synthesize the precursor pimelate moiety by use of a modified fatty acid biosynthesis pathway. However, it was previously noted that bioH has been replaced with bioG or bioK within the biotin synthetic gene clusters of other bacteria. We report that each of four BioG proteins from diverse bacteria and two cyanobacterial BioK proteins functionally replace E. coli BioH in vivo. Moreover, purified BioG proteins have esterase activity against pimeloyl-ACP methyl ester, the physiological substrate of BioH. Two of the BioG proteins block biotin synthesis when highly expressed and these toxic proteins were shown to have more promiscuous substrate specificities than the non-toxic BioG proteins. A postulated BioG-BioC fusion protein was shown to functionally replace both the BioH and BioC functions of E. coli. Although the BioH, BioG and BioK esterases catalyze a common reaction, the proteins are evolutionarily distinct.  相似文献   

8.
Escherichia coli biotin ligase can attach biotin molecules to a lysine residue of biotin acceptor peptide (BAP), and biotinylation of particular BAP-fused proteins in cells was carried out by coexpression of E. coli biotin ligase (in vivo biotinylation). This in vivo biotinylation technology has been applied for protein purification, analysis of protein localization, and protein-protein interaction in eukaryotic cells, while such studies have not been reported in bacterial cells. In this study, in vivo biotinylation of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1 was attempted by heterologous expression of E. coli biotin ligase. To biotinylate BacMPs in vivo, BAP was fused to a BacMP surface protein, Mms13, and E. coli biotin ligase was simultaneously expressed in the truncated form lacking the DNA-binding domain. This truncation-based approach permitted the growth of AMB-1 transformants when biotin ligase was heterologously expressed. In vivo biotinylation of BAP on BacMPs was confirmed using an alkaline phosphatase-conjugated antibiotin antibody. The biotinylated BAP-displaying BacMPs were then exposed to streptavidin by simple mixing. The streptavidin-binding capacity of BacMPs biotinylated in vivo was 35-fold greater than that of BacMPs biotinylated in vitro, where BAP-displaying BacMPs purified from bacterial cells were biotinylated by being mixed with E. coli biotin ligase. This study describes not only a simple method to produce biotinylated nanomagnetic particles but also a possible expansion of in vivo biotinylation technology for bacterial investigation.Biotin/streptavidin binding is the strongest noncovalent interaction known in nature (Kd [dissociation constant], ∼10−15 M) (10), and this tight binding is one of the most general tools for biological research and has been widely used for biomolecular detection (11, 12), immobilization (14, 19), and recovery (15). Therefore, it is of great significance to biotinylate biomolecules, in particular, proteins without functional inhibition. For this purpose, the method for site-selective biotinylation of proteins had been developed using biotin ligase. Biotin ligase catalyzes the posttranslational biotinylation of biotin enzymes, such as acetyl coenzyme A (acetyl-CoA) carboxylase, and introduces biotin into a specific lysine residue of a biotin carboxyl carrier protein (BCCP), a subunit of biotin enzymes (13). In early studies, BCCP (∼100 amino acid residues) had been fused with the proteins of interest for biotinylation by biotin ligase (7); however, there was a concern that fused BCCP might disrupt the function of target proteins. Recently, biotin acceptor peptides (BAPs) had replaced BCCP due to the advantage of small size. BAPs, with 15 to 23 amino acid residues, were screened from a peptide library as peptide tags biotinylated by Escherichia coli biotin ligase (4, 25). BAP-fused proteins can be biotinylated outside the cells by adding biotin and purified E. coli biotin ligase with Mg2+ and ATP (in vitro biotinylation). Furthermore, it is also possible to biotinylate BAP-fused proteins inside the cells with coexpression of E. coli biotin ligase (in vivo biotinylation) because BAP is specifically recognized only by E. coli biotin ligase. This in vivo biotinylation technology has been applied in eukaryotic cells to purify the proteins by using streptavidin-immobilized resin (8, 24, 28), because biotin/streptavidin interaction permits stringent washing to eliminate the nonspecific binding. Specific biotinylation can be applied also for protein localization analysis. Using fluorophore- or gold nanoparticle-labeled streptavidin, biotinylated proteins were clearly observed in a previous study (27). Recently, a novel technique to detect protein-protein interaction by fusing BAP and biotin ligase was developed by Ting''s group. BAP and biotin ligase were fused to different two proteins, and then the interaction of these proteins was successfully evaluated via biotinylation of BAP (9). In vivo biotinylation technology using heterologously expressed E. coli biotin ligase should be equally useful for prokaryotes; however, such studies have not been reported for bacterial cells.Magnetospirillum magneticum AMB-1, a magnetotactic bacterium, synthesizes intracellular nanosized bacterial magnetic particles (BacMPs) of 50 to 100 nm; these are surrounded by a lipid bilayer membrane, possess a single magnetic domain of magnetite, and exhibit strong ferrimagnetism (18). Furthermore, functional proteins have been displayed on BacMP surfaces through gene fusion techniques (21, 30, 31). BacMP membrane proteins, including Mms13, were used as anchor proteins; this approach permits functional proteins to be localized efficiently and oriented appropriately on BacMPs (31). We recently reported a novel method for the simple production of biotin-labeled magnetic particles through protein display techniques, where introduction of the biotin moiety onto BacMPs was carried out by the endogenous biotin ligase (17). For the biotinylation of BacMPs, we screened the gene encoding BCCP in the AMB-1 genome and displayed it on the surface of BacMPs using an anchor protein, Mms13. BCCP-displaying BacMPs were biotinylated by endogenous AMB-1 biotin ligase in the cells with high efficiency. This in vivo modification approach could be applied for construction of BacMP-quantum dot nanocomposites toward multicolor labeling of cancer cells, where BCCP and antibody carrier protein (protein G) were simultaneously displayed in tandem (16). However, the size of BCCP, with 149 amino acid residues and a mass of 15.6 kDa, makes it rather large for use as a labeling tag. Although it would be preferable to use a smaller peptide, BAP, for the tag to minimize effects on the flanking proteins for future applications, BAP was not recognized and biotinylated by endogenous AMB-1 biotin ligase (17).In this study, in vivo biotinylation of BacMPs was attempted by heterologous expression of E. coli biotin ligase and Mms13-BAP fusion protein in AMB-1 cells. First, the method for effective expression of E. coli biotin ligase in bacterial cells was optimized. Then site-selective biotinylation of BAP on BacMPs was confirmed. Finally, the obvious advantage of in vivo biotinylation of BAP-displaying BacMPs compared with the in vitro biotinylation method was demonstrated.  相似文献   

9.
Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg2+ ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.  相似文献   

10.
Glucosinolates, synthesized by the glucosinolate biosynthesis pathway, are the secondary metabolites used as a defence mechanism in the Brassicaceae plants, including Arabidopsis thaliana. The first committed step in the pathway, catalysed by methylthioalkylmalate (MAM) synthase (EC: 2.3.3.17), is to produce different variants of glucosinolates. Phylogenetic analyses suggest that possibly MAM synthases have been evolved from isopropylmalate synthase (IPMS) by the substitutions of five amino acid residues (L143I, H167L, S216G, N250G and P252G) in the active site of IPMS due to point mutations. Considering the importance of MAM synthase in Brassicaceae plants, Petersen et al. (2019) made an effort to characterise the MAM synthase (15 MAM1 variants) in vitro by single substitution or double substitutions. In their study, the authors have expressed the variants in Escherichia coli and analysed the amino acids in the cultures of E. coli in vivo. Since modifying the MAM synthases by transgenic approaches could increase the resistance of Brassicaceae plants for enhancing the defence effect of glucosinolates and their degraded products; hence, MAM synthases should be characterized in detail in vivo in A. thaliana along with the structural analysis of the enzyme for meaningful impact and for its imminent use in vivo.  相似文献   

11.
The Escherichia coli DNA mismatch repair (MMR) protein MutS is essential for the correction of DNA replication errors. In vitro, MutS exists in a dimer/tetramer equilibrium that is converted into a monomer/dimer equilibrium upon deletion of the C-terminal 53 amino acids. In vivo and in vitro data have shown that this C-terminal domain (CTD, residues 801–853) is critical for tetramerization and the function of MutS in MMR and anti-recombination. We report the expression, purification and analysis of the E.coli MutS-CTD. Secondary structure prediction and circular dichroism suggest that the CTD is folded, with an α-helical content of 30%. Based on sedimentation equilibrium and velocity analyses, MutS-CTD forms a tetramer of asymmetric shape. A single point mutation (D835R) abolishes tetramerization but not dimerization of both MutS-CTD and full-length MutS. Interestingly, the in vivo and in vitro MMR activity of MutSCF/D835R is diminished to a similar extent as a truncated MutS variant (MutS800, residues 1–800), which lacks the CTD. Moreover, the dimer-forming MutSCF/D835R has comparable DNA binding affinity with the tetramer-forming MutS, but is impaired in mismatch-dependent activation of MutH. Our data support the hypothesis that tetramerization of MutS is important but not essential for MutS function in MMR.  相似文献   

12.
Iron-sulfur (Fe-S) clusters are ubiquitous cofactors that are crucial for many physiological processes in all organisms. In Escherichia coli, assembly of Fe-S clusters depends on the activity of the iron-sulfur cluster (ISC) assembly and sulfur mobilization (SUF) apparatus. However, the underlying molecular mechanisms and the mechanisms that control Fe-S cluster biogenesis and iron homeostasis are still poorly defined. In this study, we performed a global screen to identify the factors affecting Fe-S cluster biogenesis and iron homeostasis using the Keio collection, which is a library of 3,815 single-gene E. coli knockout mutants. The approach was based on radiolabeling of the cells with [2-14C]dihydrouracil, which entirely depends on the activity of an Fe-S enzyme, dihydropyrimidine dehydrogenase. We identified 49 genes affecting Fe-S cluster biogenesis and/or iron homeostasis, including 23 genes important only under microaerobic/anaerobic conditions. This study defines key proteins associated with Fe-S cluster biogenesis and iron homeostasis, which will aid further understanding of the cellular mechanisms that coordinate the processes. In addition, we applied the [2-14C]dihydrouracil-labeling method to analyze the role of amino acid residues of an Fe-S cluster assembly scaffold (IscU) as a model of the Fe-S cluster assembly apparatus. The analysis showed that Cys37, Cys63, His105, and Cys106 are essential for the function of IscU in vivo, demonstrating the potential of the method to investigate in vivo function of proteins involved in Fe-S cluster assembly.  相似文献   

13.
The yeast Saccharomyces cerevisiae is able to use some biotin precursors for biotin biosynthesis. Insertion of a sulfur atom into desthiobiotin, the final step in the biosynthetic pathway, is catalyzed by biotin synthase (Bio2). This mitochondrial protein contains two iron-sulfur (Fe/S) clusters that catalyze the reaction and are thought to act as a sulfur donor. To identify new components of biotin metabolism, we performed a genetic screen and found that Isa2, a mitochondrial protein involved in the formation of Fe/S proteins, is necessary for the conversion of desthiobiotin to biotin. Depletion of Isa2 or the related Isa1, however, did not prevent the de novo synthesis of any of the two Fe/S centers of Bio2. In contrast, Fe/S cluster assembly on Bio2 strongly depended on the Isu1 and Isu2 proteins. Both isa mutants contained low levels of Bio2. This phenotype was also found in other mutants impaired in mitochondrial Fe/S protein assembly and in wild-type cells grown under iron limitation. Low Bio2 levels, however, did not cause the inability of isa mutants to utilize desthiobiotin, since this defect was not cured by overexpression of BIO2. Thus, the Isa proteins are crucial for the in vivo function of biotin synthase but not for the de novo synthesis of its Fe/S clusters. Our data demonstrate that the Isa proteins are essential for the catalytic activity of Bio2 in vivo.  相似文献   

14.
An oxygen requirement for de novo biotin synthesis in Saccharomyces cerevisiae precludes the application of biotin-prototrophic strains in anoxic processes that use biotin-free media. To overcome this issue, this study explores introduction of the oxygen-independent Escherichia coli biotin-biosynthesis pathway in S. cerevisiae. Implementation of this pathway required expression of seven E. coli genes involved in fatty-acid synthesis and three E. coli genes essential for the formation of a pimelate thioester, key precursor of biotin synthesis. A yeast strain expressing these genes readily grew in biotin-free medium, irrespective of the presence of oxygen. However, the engineered strain exhibited specific growth rates 25% lower in biotin-free media than in biotin-supplemented media. Following adaptive laboratory evolution in anoxic cultures, evolved cell lines that no longer showed this growth difference in controlled bioreactors, were characterized by genome sequencing and proteome analyses. The evolved isolates exhibited a whole-genome duplication accompanied with an alteration in the relative gene dosages of biosynthetic pathway genes. These alterations resulted in a reduced abundance of the enzymes catalyzing the first three steps of the E. coli biotin pathway. The evolved pathway configuration was reverse engineered in the diploid industrial S. cerevisiae strain Ethanol Red. The resulting strain grew at nearly the same rate in biotin-supplemented and biotin-free media non-controlled batches performed in an anaerobic chamber. This study established an unique genetic engineering strategy to enable biotin-independent anoxic growth of S. cerevisiae and demonstrated its portability in industrial strain backgrounds.  相似文献   

15.
InhA, the primary target for the first line anti-tuberculosis drug isoniazid, is a key enzyme of the fatty-acid synthase II system involved in mycolic acid biosynthesis in Mycobacterium tuberculosis. In this study, we show that InhA is a substrate for mycobacterial serine/threonine protein kinases. Using a novel approach to validate phosphorylation of a substrate by multiple kinases in a surrogate host (Escherichia coli), we have demonstrated efficient phosphorylation of InhA by PknA, PknB, and PknH, and to a lower extent by PknF. Additionally, the sites targeted by PknA/PknB have been identified and shown to be predominantly located at the C terminus of InhA. Results demonstrate in vivo phosphorylation of InhA in mycobacteria and validate Thr-266 as one of the key sites of phosphorylation. Significantly, our studies reveal that the phosphorylation of InhA by kinases modulates its biochemical activity, with phosphorylation resulting in decreased enzymatic activity. Co-expression of kinase and InhA alters the growth dynamics of Mycobacterium smegmatis, suggesting that InhA phosphorylation in vivo is an important event in regulating its activity. An InhA-T266E mutant, which mimics constitutive phosphorylation, is unable to rescue an M. smegmatis conditional inhA gene replacement mutant, emphasizing the critical role of Thr-266 in mediating post-translational regulation of InhA activity. The involvement of various serine/threonine kinases in modulating the activity of a number of enzymes of the mycolic acid synthesis pathway, including InhA, accentuates the intricacies of mycobacterial signaling networks in parallel with the changing environment.  相似文献   

16.
Metabolic engineering is used to improve titers, yields and generation rates for biochemical products in host microbes such as Escherichia coli. A wide range of biochemicals are derived from the central carbon metabolite acetyl-CoA, and the largest native drain of acetyl-CoA in most microbes including E. coli is entry into the tricarboxylic acid (TCA) cycle via citrate synthase (coded by the gltA gene). Since the pathway to any biochemical derived from acetyl-CoA must ultimately compete with citrate synthase, a reduction in citrate synthase activity should facilitate the increased formation of products derived from acetyl-CoA. To test this hypothesis, we integrated into E. coli C ΔpoxB twenty-eight citrate synthase variants having specific point mutations that were anticipated to reduce citrate synthase activity. These variants were assessed in shake flasks for growth and the production of acetate, a model product derived from acetyl-CoA. Mutations in citrate synthase at residues W260, A267 and V361 resulted in the greatest acetate yields (approximately 0.24 g/g glucose) compared to the native citrate synthase (0.05 g/g). These variants were further examined in controlled batch and continuous processes. The results provide important insights on improving the production of compounds derived from acetyl-CoA.  相似文献   

17.
Recent work implicated the Escherichia coli BioC protein as the initiator of the synthetic pathway that forms the pimeloyl moiety of biotin (Lin, S., Hanson, R. E., and Cronan, J. E. (2010) Nat. Chem. Biol. 6, 682–688). BioC was believed to be an O-methyltransferase that methylated the free carboxyl of either malonyl-CoA or malonyl-acyl carrier protein based on the ability of O-methylated (but not unmethylated) precursors to bypass the BioC requirement for biotin synthesis both in vivo and in vitro. However, only indirect proof of the hypothesized enzymatic activity was obtained because the activities of the available BioC preparations were too low for direct enzymatic assay. Because E. coli BioC protein was extremely recalcitrant to purification in an active form, BioC homologues of other bacteria were tested. We report that the native form of Bacillus cereus ATCC10987 BioC functionally replaced E. coli BioC in vivo, and the protein could be expressed in soluble form and purified to homogeneity. In disagreement with prior scenarios that favored malonyl-CoA as the methyl acceptor, malonyl-acyl carrier protein was a far better acceptor of methyl groups from S-adenosyl-l-methionine than was malonyl-CoA. BioC was specific for the malonyl moiety and was inhibited by S-adenosyl-l-homocysteine and sinefungin. High level expression of B. cereus BioC in E. coli blocked cell growth and fatty acid synthesis.  相似文献   

18.
Hexameric DnaB type replicative helicases are essential for DNA strand unwinding along with the direction of replication fork movement. These helicases in general contain an amino terminal domain and a carboxy terminal domain separated by a linker region. Due to the lack of crystal structure of a full-length DnaB like helicase, the domain structure and function of these types of helicases are not clear. We have reported recently that Helicobacter pylori DnaB helicase is a replicative helicase in vitro and it can bypass Escherichia coli DnaC activity in vivo. Using biochemical, biophysical and genetic complementation assays, here we show that though the N-terminal region of HpDnaB is required for conformational changes between C6 and C3 rotational symmetry, it is not essential for in vitro helicase activity and in vivo function of the protein. Instead, an extreme carboxy terminal region and an adjacent unique 34 amino acid insertion region were found to be essential for HpDnaB activity suggesting that these regions are important for proper folding and oligomerization of this protein. These results confer great potential in understanding the domain structures of DnaB type helicases and their related function.  相似文献   

19.
Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS–DHFR specific inhibitors.  相似文献   

20.
Biotin synthase (BioB) is an S-adenosylmethionine radical enzyme that catalyzes addition of sulfur to dethiobiotin to form the biotin thiophane ring. In vitro, Escherichia coli BioB is active for only one turnover, during which the [2Fe-2S]2+ cluster is destroyed, one sulfide from the cluster is incorporated as the biotin thiophane sulfur, while Fe2+ ions and the remaining S2− ion are released from the protein. The present work examines the fate of the protein following the loss of the FeS clusters. We examine the quaternary structure and thermal stability of active and inactive states of BioB, and find that loss of either the [4Fe-4S]2+ or [2Fe-2S]2+ clusters results in destabilization but not global unfolding of BioB. Using susceptibility to limited proteolysis as a guide, we find that specific regions of the protein appear to be transiently unfolded following loss of these clusters. We also examine the in vivo degradation of biotin synthase during growth in low-iron minimal media and find that BioB is degraded by an apparent ATP-dependent proteolysis mechanism that sequentially cleaves small fragments starting at the C-terminus. BioB appears to be resistant to degradation and capable of multiple turnovers only under high-iron conditions that favor repair of the FeS clusters, a process most likely mediated by the Isc or Suf iron-sulfur cluster assembly systems.  相似文献   

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