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Characterization of a Human Peptide Deformylase:  Implications for Antibacterial Drug Design †

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Characterization of a Human Peptide Deformylase:  Implications for Antibacterial Drug Design †
  Characterization of a Human Peptide Deformylase: Implications for AntibacterialDrug Design † Kiet T. Nguyen, ‡,§ Xubo Hu, ‡ Craig Colton, §, | Ratna Chakrabarti, ⊥ Michael X. Zhu, §, | and Dehua Pei* ,‡,§  Department of Chemistry, Neuroscience and Neurobiotechnology Center, and Ohio State Biochemistry Program,The Ohio State Uni V  ersity, 100 West 18th A V  enue, Columbus, Ohio 43210, and Department of Molecular Biology and  Microbiology, Uni V  ersity of Central Florida, 12722 Research Parkway, Orlando, Florida 32826  Recei V  ed April 23, 2003; Re V  ised Manuscript Recei V  ed June 25, 2003 ABSTRACT : Ribosomal protein synthesis in eubacteria and eukaryotic organelles initiates with an  N  -formylmethionyl-tRNA i , resulting in N-terminal formylation of all nascent polypeptides. Peptidedeformylase (PDF) catalyzes the subsequent removal of the N-terminal formyl group from the majorityof bacterial proteins. Deformylation was for a long time thought to be a feature unique to the prokaryotes,making PDF an attractive target for designing novel antibiotics. However, recent genomic sequencinghas revealed PDF-like sequences in many eukaryotes, including man. In this work, the cDNA encoding  Homo sapiens  PDF (HsPDF) has been cloned and a truncated form that lacks the N-terminal 58-amino-acid targeting sequence was overexpressed in  Escherichia coli . The recombinant, Co 2 + -substituted proteinis catalytically active in deformylating N-formylated peptides, shares many of the properties of bacterialPDF, and is strongly inhibited by specific PDF inhibitors. Expression of HsPDF fused to the enhancedgreen fluorescence protein in human embryonic kidney cells revealed its location in the mitochondrion.However, HsPDF is much less active than its bacterial counterpart, providing a possible explanation forthe apparent lack of deformylation in the mammalian mitochondria. The lower catalytic activity is atleast partially due to mutation of a highly conserved residue (Leu-91 in  E. coli  PDF) in mammalian PDF.PDF inhibitors had no detectable effect on two different human cell lines. These results suggest thatHsPDF is likely an evolutional remnant without any functional role in protein formylation/deformylationand validates PDF as an excellent target for antibacterial drug design. In prokaryotes and eukaryotic organelles (e.g., mitochon-dria and plastids), ribosomal protein biosynthesis is initiatedwith N-formylmethionine ( 1 ). Consequently, all nascentpolypeptides synthesized in bacteria, mitochondria, andchloroplasts bear an N-terminal formyl group. In bacteria,the N-formyl group is subsequently removed from the vastmajority of polypeptides by peptide deformylase (PDF), 1 apparently as a co-translational event ( 2 - 4 ). A fraction of the deformylated polypeptides undergo further N-terminalprocessing (i.e., removal of N-terminal methionine bymethionine aminopeptidase) to give mature proteins ( 1 ). PDFis a unique and highly unstable metallopeptidase, whichcontains a ferrous ion (Fe 2 + ) as the catalytic metal ( 5 ,  6  ).PDF is essential for bacterial survival; either deletion of the def   gene ( 7  - 9 ) or treatment with a PDF inhibitor ( 10 - 13 )prevents bacterial growth. These properties make PDF anattractive target for developing novel antibiotics, and one of the PDF inhibitors is currently in phase I clinical trials forthe treatment of upper respiratory tract infections.Another factor that made PDF an attractive antibacterialdrug target was the common belief, in the past, that theenzyme was unique to the bacterial kingdom. Indeed,cytoplasmic protein synthesis in eukaryotes does not involveN-formylation, and therefore, there is no need for deformy-lation ( 1 ). Available evidence also suggests that there is nodeformylation in the mitochondrion of mammals. Forexample, the intramitochondrially synthesized proteins frombovine and rat typically retain their N-terminal formyl groupor have their N-terminal signal sequences removed (reviewedin refs  2 - 4 ). However, genomic sequencing has recentlyrevealed many PDF-like sequences in parasites, plants, andmammals ( 2 - 4 ). Meinnel and co-workers have shown thatthe two  def  -like genes in  Arabidopsis thaliana  indeed codefor functional PDFs ( 14 ). We previously reported the cloningand characterization of an eukaryotic PDF from the malariaparasite,  Plasmodium falciparum  ( 15 ). The presence of PDF-like sequences in the mammalian genome raises severalimportant questions such as the function of PDF in mammalsand the suitability of PDF as a novel drug target. To beginto address these questions, we have undertaken the cloningand characterization of human PDF (HsPDF). Biochemical † This work was supported by a grant from the National Institutesof Health (AI40575 to D.P.).* Corresponding author. Address: Department of Chemistry, TheOhio State University, 100 West 18th Avenue, Columbus, OH 43210.Telephone: (614) 688-4068. Fax: (614) 292-1532. E-mail: ‡ Department of Chemistry, The Ohio State University. § Ohio State Biochemistry Program, The Ohio State University. | Neuroscience and Neurobiotechnology Center, The Ohio StateUniversity. ⊥ University of Central Florida. 1 Abbreviations: PDF, peptide deformylase; HsPDF,  Homo sapiens PDF; EcPDF,  E. coli  PDF; PfPDF,  Plasmodium falciparum  PDF; GFP,green fluorescence protein; HEK, human embryonic kidney; GST,glutathione-S-transferase. 9952  Biochemistry  2003,  42,  9952 - 9958 10.1021/bi0346446 CCC: $25.00 © 2003 American Chemical SocietyPublished on Web 07/30/2003  and kinetic analyses show that the recombinant protein isan active PDF, although its activity is considerably lowerthan that of the bacterial enzyme. In addition, known PDFinhibitors inhibit its catalytic activity in vitro but had littleeffect on the growth of human cells. The results are consistentwith the hypothesis that HsPDF is a mere evolutionalremnant of no current function. MATERIALS AND METHODS  Materials.  All Fmoc-protected amino acids, 2-(1  H  -ben-zotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophos-phate (HBTU), and 1-hydroxybenzotriazole (HOBT) werepurchased from SynPep (Dublin, CA). Rink resin was fromAdvanced Chemtech (Louisville, KY). Formate dehydroge-nase was from Sigma.  Aeromonas  aminopeptidase waspurified as previously described ( 16  ). Other chemicals,including isopropyl-   - D -thiogalactopyranoside (IPTG), phe-nylmethanesulfonyl fluoride, kanamycin, ethanedithiol andits derivatives, and   -mercaptoethanol, were purchased fromAldrich.  Buffers.  Buffer A, 50 mM Hepes (pH 7.2), 100 mM NaCl,1 mM   -mercaptoethanol, and 1% Triton X-100; buffer B,20 mM Hepes (pH 7.2), 100 mM NaCl; buffer C, 50 mMTris ‚ HCl, 10 mM NaCl, pH 8.0; buffer D, 50 mM Tris ‚ HCl(pH 8.0), 1 mM NaCl; buffer E, 50 mM Hepes (pH 7.0),150 mM NaCl; buffer F, 20 mM Hepes (pH 7.2), 100 mMNaCl, 0.2 mM   -mercaptoethanol; buffer G, 50 mM Mops(pH 7.0), 10 mM NaCl. Cloning, Expression, and Purification of HsPDF.  On thebasis of the reported HsPDF genomic DNA sequence at, two polymerase chain reaction(PCR) primers were designed as follows: 5 ′ -GGGGATC-CATATGGCCCGGCTGTGGGGCGCGCTGAGTCTT-3 ′  and5 ′ -GGGAATTCTTAGTCATTCACCTTCATCCAATAG-3 ′ . Approximately 0.1  µ g of a human fetus Marathon-ReadycDNA library (Clontech, CA) was used in the PCR reaction.The Clontech Advantage-GC PCR mixture was employedto destabilize any secondary structures in G,C-rich DNA ( 17  ).The PCR reaction (50  µ L total volume) contained 800  µ MdNTPs, 0.2  µ M of each primer, 1.0 M GC melt mixture,and 1  µ L of 50 × Advantage-GC 2 polymerase mix. The PCRwas performed for 32 cycles with the following conditions:94  ° C for 30 s/52  ° C for 60 s/68  ° C for 70 s. The 0.73 kbPCR product was purified on a Qiaquick column (Qiagen)to remove any free nucleotides, digested with restrictionendonucleases  Nde I and  Xho I, and cloned into the prokaryoticexpression vector pET-22b (Novagen, WI) to produceplasmid pET22b-HsPDF. The entire coding region of HsPDFwas sequenced and found to be identical to the publishedsequence.N-terminal truncation of HsPDF was carried out by PCRusing the above 3 ′  primer and the following 5 ′  primers: 5 ′ -GGCCCATGGAACGGCGCTCCTATTGGCGCCA-3 ′ , 5 ′ -GGCCCATGGAACCTGAGGCGTCTGGTGCTGG-3 ′ , and5 ′ -GGCCCATGGAACCTCCCGAACCGCCGTTCTCG-3 ′ . These primers resulted in the truncation of 44, 52, and58 amino acids, respectively, from the N-terminus of HsPDF.The PCR products were cloned into the plasmid pET-42bto generate plasmids pET42b-HsPDF ∆ 44, pET42b-HsP-DF ∆ 52, and pET42b-HsPDF ∆ 58, respectively. This cloningprocedure resulted in the in-frame addition of an N-terminalglutathione-S-transferase (GST) tag along with a Factor Xacleavage site.  E. coli  BL21 (DE3) Rosetta cells (Novagen) carrying theproper plasmid were grown in minimal media containing 60  µ g/mL kanamycin and 35  µ g/mL chloramphenicol at 37  ° Cuntil OD 600  reached  ∼ 0.9. The culture was supplementedwith 100  µ M CoCl 2  and 100  µ M isopropyl-   - D -thiogalac-topyranoside and incubated at 30  ° C for an additional 3 h.The cells (12 L) were chilled on ice for 1 h and harvestedby centrifugation. The cell pellet was suspended in 200 mLof buffer A plus 50  µ g/mL phenylmethanesulfonyl fluoride,0.5% protamine sulfate, 20  µ g/mL trypsin inhibitor, and 100  µ g/mL lysozyme. The mixture was stirred at 4  ° C for 30min and briefly sonicated (5 × 10 s pulses). The crude lysatewas centrifuged to yield a clear supernatant, which wasmixed with 10 mL of GST bind resin (Pharmacia). Thecolumn was washed with 300 mL of buffer F and elutedwith 50 mL of buffer F containing 0.8 mM reducedglutathione. The GST - HsPDF fractions were pooled andconcentrated to  ∼ 2 mL using an Amicon YM-3 cellulosemembrane filter. The resulting solution was passed througha Pharmacia FPLC Fast-desalting column (eluted with bufferC) to remove the gluthathione and salts. Fractions containingGST - HsPDF were pooled ( ∼ 3 mL) and treated with 30 unitsof Factor Xa (Novagen) at 4  ° C for 8 h to cleave the GSTtag. HsPDF was purified by passing through a monoQ HR5/5 anion-exchange column equilibrated in buffer C. Thecolumn was eluted with buffer C plus a linear gradient of 10 - 1000 mM NaCl. Fractions containing HsPDF werepooled and concentrated in an Amicon YM-3 cellulose filter.Protein concentration was determined by Bradford assayusing bovine serum albumin as the standard. Typically, ∼ 1mg of pure HsPDF was obtained from a 12-L culture. Metalanalysis was performed for the GST - HsPDF fusion proteinby inductively coupled plasma emission spectroscopy (ICP-ES) at the Chemical Analysis Laboratory of the Universityof Georgia. Site-Directed Mutagenesis.  Mutation of Glu-173 to leucinein HsPDF was performed by the Quick-Change mutagenesismethod using the following primers: 5 ′ -CCGAGGGCTG-CGCTAGCGTCGCCGGCT-3 ′  and 5 ′ -AGCCGGCGACG-CTAGCGCAGCCCTCGG-3. The DNA amplification reac-tion contained 800  µ M dNTPs, 0.1  µ g of the plasmidpET42b-HsPDF ∆ 58 DNA, 0.2  µ M of each primer, 1.0 MGC melt mixture, and 2.5 units of   Pfu Turbo DNA poly-merase (Stratagene). Twenty cycles were performed asfollows: 95  ° C for 30 s/54  ° C for 60 s/72  ° C for 14 min.The identity of the mutants was confirmed by DNA sequenc-ing. The mutant was expressed and purified in the samemanner as the wild-type enzyme. Mutation of Leu-91 of EcPDF to a glutamate was similarly carried out with thefollowing primer pair: 5 ′ -GAAGAAGGTTGCGAGTC-GATCCCTGAACAACGTG-3 ′  and 5 ′ -CACGTTGTTCAGG-GATCGACTCGCAACCTTCTTC-3 ′ , except that the plas-mid pET22b-EcPDF ( 18  ) was used as the template. Expressionand purification of L91E EcPDF were carried out aspreviously described ( 19 ). Peptide Synthesis.  All peptides were prepared by solid-phase synthesis on Rink resin as previously described ( 18  ).HPLC analysis showed generally > 85% purity. The identityof the peptides was confirmed by matrix-assisted laserdesorption ionization mass spectrometry.Deformylase as Drug Target  Biochemistry, Vol. 42, No. 33, 2003  9953  Synthesis of PDF Inhibitors . PDF inhibitors  1 - 4  weresynthesized from commercially available starting materials.The synthetic details are described in the SupportingInformation. PDF Assays.  Two different methods were employed toassay for PDF activity. Method A employed f-ML-pNA assubstrate which, upon deformylation by PDF, is furtherprocessed by  Aeromonas  aminopeptidase (AAP) to release  p -nitroaniline ( 20 ). The assay reaction (total volume of 1.0mL) typically contained buffer E, 1 mM tris(2-carboxyethyl)-phosphine (TCEP), 0 - 200  µ M f-ML-pNA, and 2 units of AAP. The reaction was initiated by the addition of 1 - 10  µ g of HsPDF, and the reaction progress was monitored at405 nm on a UV - vis spectrophotometer. The initial reactionrate was calculated from the early, linear region of theprogress curve (0 - 45 s). Method B was used for all otherN-formylated substrates. It couples the PDF reaction withformate dehydrogenase, which oxidizes formate into carbondioxide while reducing NAD + to NADH ( 18  ,  21 ). A typicalreaction (total volume of 500  µ L) contained buffer G, 1 mMTCEP, and 0 - 2 mM N-formylated peptide. The reaction wasinitiated by the addition of 1 - 10  µ g of HsPDF, and allowedto proceed for 30 min at room temperature before beingquenched by heating at 95  ° C for 10 min (the inactivationprocess is usually complete within the first 30 s). Aftercooling to room temperature, the amount of released formatewas quantified as previously described ( 18  ,  21 ). Inhibitionassays (method A) were carried out with 150  µ M f-ML-pNA as substrate, 0 - 400  µ M inhibitor, and 1.1  µ g of HsPDF.The reaction was quenched by heating at 95  ° C for 10 minand cooled to room temperature. After the addition of AAP(1.0 unit) and incubation for 15 min, the absorbance increaseat 405 nm was measured. For all end-point assay reactions,the substrate to product conversion was kept at  < 20%. Fluorescence Microscopy.  A PCR was performed withplasmid pET22b-HsPDF as template and primers 5 ′ -TTGCTCGAGATGGCCCGGCTGTGGGGCGCGC-3 ′  and5 ′ -GCCGGATCCTCATTCACCTTCATCCAATA-3 ′ . Theresulting full-length HsPDF cDNA was digested with  Xho Iand  Bam HI and cloned into a mammalian expression vectorpEGFP(N1) (Clontech). This cloning procedure resulted inthe in-frame fusion of the enhanced green fluorescenceprotein (EGFP) at the C-terminus of HsPDF to produce theplasmid pEGFP-HsPDF. Approximately 800 000 humanembryonic kidney (HEK293) cells in 2 mL of Dulbecco’sminimum essential medium (DMEM) with high glucosecontaining 10% fetal bovine serum (FBS) were plated ontoa sterile 35 mm plastic plate and incubated at 37  ° Covernight. Transient transfection of pEGFP - HsPDF intoHEK cells was performed according to LipofectAMINE 2000protocol (Life Technologies). The transfected cells wereincubated at 37  ° C in a CO 2  incubator for 48 h and weresubsequently trypsinized and plated onto glass cover slips.After 24 h, the cells were stained with MitoTracker RedCMXRos (Molecular Probes), and the glass cover slip wasremoved with the adhered HEK cells. Cells were fixed with4% paraformaldehyde. Visualization was performed undera Bio-Rad MRC-1024 confocal laser scanning unit equippedwith a krypton/argon laser, a photomultiplier tube, and anupright Nikon microscope. Images were taken with a 60 × oil objective at an Iris setting of 3. The 488 and 568 nmlaser line was used separately to acquire images for the GFPand MitoTracker, respectively. Merged images were gener-ated using the Confocal Assistant software (Bio-Rad). Cell Growth Inhibition Assay.  To facilitate the monitoringof growth rate using a fluorescence plate reader, HEK 293cells were transfected with the mammalian expression vectorpEGFP-IRESneo (Clontech) as described above. Cells weregrown on a 60 mm polystyrene plate with 4 mL of DMEM +  FBS media supplemented with 400  µ g/mL G418. Tomaintain a stable cell line, the transfected cell line was grownto  > 95% confluency (typically 3 days), trypsinized, andsubcultured for 8 weeks. To test the effect of PDF inhibitors,cells were plated onto a polyornithine-treated 96-wellpolystyrene plate in 150  µ L of DMEM + FBS ( ∼ 20 000 cellsper well), and grown overnight. The media was removedand replaced with fresh DMEM + FBS containing 0 - 128  µ MPDF inhibitor  1 . Fluorescence signal was measured every 8h over a 3 day period using the FLEXstation (MolecularDevices) at an excitation wavelength of 485 nm and anemission wavelength of 525 nm at 23  ° C.  DNA Synthesis Inhibition Assay . The cell line used,L5178YD10/R cells, is an acute lymphoblastic leukemia cellline resistant to  L -asparaginase (obtained from ATCC). Cellswere maintained in DMEM with high-glucose-containing10% FBS, 1% antibiotic/antimycotic and 1%  L -glutamine.Cells in log phase were seeded in a 96-well plate at a dilutionof 5 × 10 5 cell/200  µ L in three wells, which was served asthe negative control. Log phase cells at a concentration of 2.5  ×  10 6 cells /ml were then mixed with  3 H-thymidine (1  µ Ci/mL of cell suspension) and added to the wells (200  µ L/ well). Cells in individual wells were treated with PDFinhibitor 1 or 2 at the specified concentrations (in triplicate)ranging from 50 nM to 150  µ M and incubated at 37  ° C for18 h in a CO 2  incubator. At the end of incubation, cells wereharvested in a cell harvester (Tomech Harvester), and labeledDNA was captured on filter membranes. Membrane-boundDNA was washed in distilled water and air-dried for 3 h.Radioactivity retained on the membrane was measured in aliquid scintillation counter (Beckman). RESULTS O V  erexpression and Purification of HsPDF.  A BLASTsearch of the human genomic sequence stored at the NCBIwebsite ( with EcPDF se-quence as query resulted in a single PDF-like homologue,located on chromosome 16 (GenBank accession numberAF23915). On the basis of the predicted protein sequence( 14 ), we cloned the full-length cDNA, which encodes aprotein of 243 amino acids, from a human fetal tissue cDNAlibrary by PCR. Sequence alignment shows that HsPDFcontains a catalytic domain, which shares  ∼ 30% sequenceidentity to EcPDF, and a 61-amino acid extension at theN-terminus (Figure 1). The N-terminal extension resemblesthat of   P. falciparum  PDF (PfPDF) ( 15 ). We anticipated thatthe full-length HsPDF would be difficult to express as asoluble protein in  E. coli ; therefore, we constructed varioustruncation mutants that lack the N-terminal 44 ( ∆ N44), 52( ∆ N52), and 58 ( ∆ N58) amino acids. We first cloned thetruncation mutants in prokaryotic expression vector pET22band attempted to overexpress them in  E. coli  BL21(DE3)cells. Unfortunately, neither the mutants nor the full-lengthprotein could be expressed at significant levels. After9954  Biochemistry, Vol. 42, No. 33, 2003  Nguyen et al.  considering the different codon bias between human andbacterial systems, we next constructed HsPDF as a GSTfusion protein and employed an  E. coli  Rosetta strain thatcarries several rare tRNA’s normally found in  E. coli . TheGST fusion would also facilitate its purification on aglutathione column.Previous studies have shown that bacterial PDF containsa catalytic Fe 2 + ion and is highly sensitive to molecularoxygen and/or H 2 O 2  in solution ( 22 ). Replacing the ferrousion with Co 2 + or Ni 2 + results in a stable enzyme of essentiallywild-type activity ( 6  ,  19 ,  23 ). Therefore, we produced HsPDFin the Co 2 + -substituted form by growing  E. coli  cells inminimal medium supplemented with 100  µ M CoCl 2 . The ∆ N44 and  ∆ N52 variants did not produce any detectableprotein on SDS - PAGE gels. However, we were able toexpress the  ∆ N58 variant and partially purify it on aglutathione column. The  ∆ N58 protein thus obtained stillcontained multiple bands, most of them smaller than the 45-kDa GST - ∆ N58 fusion protein (Figure 2, lane 4). Webelieve that the smaller species are due to proteolysis of theHsPDF portion of the fusion. Consistent with this notion,when cells were induced for  > 6 h, the intensity of the 45-kDa band actually decreased (data not shown). Metal analysisof the GST - HsPDF fusion revealed that the protein con-tained approximately 0.67 mol of Co 2 + and 0.33 mol of Zn 2 + ,but no significant amounts of any other divalent metals.Cleavage of the GST fusion with protease Factor Xa,followed by anion-exchange chromatography, producedCo 2 + -HsPDF of apparent homogeneity (Figure 2, lane 6).Using the above procedure, ∼ 1.0 mg of HsPDF was obtainedfrom 12 L of   E. coli  cells. Catalytic Properties of HsPDF.  We first evaluated thecatalytic properties of HsPDF against an artificial substrate,f-ML-pNA. HsPDF is a catalytically active enzyme, with a k  cat  of 0.17 s - 1 , a  K  M  of 27  µ M, and a  k  cat  /  K  M  of 5.9  ×  10 3 M - 1 s - 1 (Table 1). This activity is comparable to that of another eukaryotic PDF, PfPDF, but is ∼ 200-fold lower thanthat of EcPDF ( 15 ,  19 ). Since HsPDF is located in the humanmitochondrion (vide infra), its physiological substrates, if any, would be the mitochondrially synthesized proteins. Thehuman mitochondrial DNA encodes 13 proteins ( 24 ). Wesynthesized six N-formylated hexapeptides corresponding tothe N-terminal sequences of six of the human mitochondrial F IGURE  1: Sequence alignment of various PDF’s from eukaryotic and prokaryotic organisms:  Plasmodium Falciparum  ( Pf  ),  Escherichiacoli  (  Ec ),  Homo sapiens  (  Hs ),  Bacillus subtilis  (  Bs ),  Staphylococcus aureus  ( Sa ), and  Arabidopsis thaliana  (  At  ). Alignment was performedby inputting PDF sequences into ClustalW software located at website.F IGURE  2: A 15% SDS - PAGE gel showing the purity of HsPDF-( ∆ N58) during different stages of purification. Lane 1, molecular-weight markers; lane 2, crude cell lysate; lane 3, flow-throughfraction of the GST-bind column; lane 4, proteins eluted from theGST-bind column; lane 5, after factor Xa cleavage; and lane 6,HsPDF after purification on Mono-Q column. Molecular weightstandards (kD) are indicated on the left side of the gel. Deformylase as Drug Target  Biochemistry, Vol. 42, No. 33, 2003  9955  proteins (cytochrome  c  oxidase II and III, NADH dehydro-genase subunit II and V, ATP synthase F0 subunit 8, andcytochrome  b ) (Table 1). HsPDF exhibit low but detectableactivity to three of the peptides (f-MAHAAQ, f-MTHQSH,and f-MTMHTT), with  k  cat  /  K  M  values in the range of 200 - 800 M - 1 s - 1 . These activities are again 20 - 50-fold lower thanthose of EcPDF against the same substrates (Table 2). Twoof the peptides had very poor solubility and accurate activitymeasurement was not possible for either EcPDF or HsPDF(f-MNPLAQ and f-MPQKNT). HsPDF showed no detectableactivity toward the remaining peptide (f-MTPMRK).A striking difference between HsPDF (also mouse PDF)and bacterial PDF is that the leucine in the highly conservedEGCLS motif is mutated into a glutamic acid in mammalianPDF (Glu-173 in HsPDF). The leucyl side chain is involvedin hydrophobic interactions with the P 2 ′  side chain of asubstrate ( 25 - 27  ). To determine whether this mutation isresponsible for the low activity of HsPDF, we mutated Glu-173 of HsPDF back to leucine and the corresponding leucine(Leu-91) in EcPDF to a glutamic acid. When assayed againstthe above set of substrates, the E173L mutant HsPDF showedconsistently higher activity than the wild-type enzyme (2 - 4-fold). Vice versa, mutation of Leu-91 to Glu resulted in a100-fold reduction of catalytic activity in EcPDF (Table 2).Thus, mutation of the conserved leucyl residue is at leastpartially responsible for the low catalytic activity of mam-malian PDF.  Intracellular Localization of HsPDF.  The function of theN-terminal extension of HsPDF was assessed by severalprediction software, including signalP ( 28  ) and Predotar( Both programs pre-dicted that the N-terminal sequence contain organelle target-ing signals. Also, since the mitochondrion is the only placewhere N-formylated proteins are synthesized, one wouldexpect that HsPDF be localized in the mitochondrion, if itsfunction were to deformylate proteins. To determine thesubcellular localization of HsPDF, we fused the greenfluorescence protein to the C-terminus of the full-lengthHsPDF. The fusion protein was transiently expressed in HEKcells. Confocal laser scanning microscopy revealed that thefusion protein is localized in the mitochondria (Figure 3).As a control, we transfected the HEK cells with theexpression vector pEGFP(N1) alone, which produces onlyGFP in the cells. Green fluorescence was observed through-out the cytoplasm (data not shown).  Inhibition of HsPDF.  Since PDF is being pursued as atarget for antibacterial drug design, an important issue iswhether the inhibitors designed against bacterial PDF willalso inhibit HsPDF. BB-3497 (compound  1  in Table 3) is apotent inhibitor against bacterial PDF ( K  I  )  7 nM againstEcPDF) and an orally available, broad-spectrum antibacterialagent ( 13 ). Its N-formylhydroxylamine moiety binds to thecatalytic metal of PDF in a bidentate fashion ( 13 ). To addressthe issue, we synthesized BB-3497 and three analogues(compounds  2 - 4  in Table 3) and tested them against bothHsPDF and EcPDF. All of the compounds showed potentinhibition of both enzymes, with  K  I  values in the low tomedium nM range (Table 3). Among them, BB-3497 is themost potent, showing competitive inhibition pattern (Figure4) and  K  I  values of 8 and 11 nM (this work) against HsPDFand EcPDF, respectively. Further, with the exception of  Table 1: HsPDF Activity against f-ML-pNA and HumanMitochondrial Peptidessubstrate k  cat (s - 1 ) K  M (  µ M) k  cat  /  K  M (M - 1 s - 1 )f-ML-pNA 0.17 ( 0.06 27 ( 6 5900f-MAHAAQ a 0.57 ( 0.08 2500 ( 310 230f-MTHQSH b 1.5 ( 0.5 1850 ( 230 810f-MNPLAQ c ND ND NDf-MTMHTT d  2.2 ( 0.9 4000 ( 800 545f-MPQLNT e ND ND NDf-MTPMRK  f  NA NA NA a These peptides correspond to the N-terminal sequences of humanmitochondrial proteins:  a , cytochrome  c  oxidase II;  b , cytochrome  c oxidase III;  c , NADH dehydrogenase subunit II;  d  , NADH dehydro-genase subunit V;  e , ATP synthase F0 subunit 8; and  f  , cytochrome  b .ND, activity not determined due to insolubility. NA, no detectableactivity. All assays were performed in triplicates and data reportedrepresent the mean  (  SD.Table 2: Catalytic Activity of Wild-Type vs Mutant HsPDF and EcPDF k  cat  /  K  M  (M - 1 s - 1 )substrate Wt HsPDF E173L HsPDF Wt EcPDF L91E EcPDFf-ML-pNA 5900 ( 1100 21800 ( 3000 (1.2 ( 0.1) × 10 6 12000 ( 2000f-MAHAAQ 230 ( 55 810 ( 60 12000 ( 1600 NDf-MTHQSH 810 ( 65 1200 ( 400 15000 ( 1300 NDf-MTMHTT 540 ( 50 1320 ( 450 29000 ( 5800 NDf-MTPMRK NA NA 2390 ( 230 NDND, not determined; NA, no detectable activity. F IGURE  3: Intracellular localization of HsPDF - GFP fusion in HEK cells. Fluorescence signal was visualized under a confocal microscope.(A) Fluorescence signal of mitochondria stained by MitoTracker (red). (B) Fluorescence signal of GFP (green). (C) An overlay of panelsA and B. 9956  Biochemistry, Vol. 42, No. 33, 2003  Nguyen et al.
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