Identification of amino acid residues involved in the activity of ...

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THEJOURNALOF BIOUXICALCHEMISTRY 0 1994 by The American Soeiety for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 7, Issue of February 18, pp. 4872-4877, 1994 Printed in U S A .

Identification of Amino AcidResidues Involvedin theActivity of Phosphomannose Isomerase-Guanosine5’-Diphospho-~-mannose Pyrophosphorylase A BIFUNCTIONAL ENZYME IN THE ALGINATE BIOSYNTHETIC PATHWAY OF PSEUDOMONAS AERUGINOSA* (Received for publication, March 23, 1993, and in revised form, September 23, 1993)

Thomas B. May$, Dean Shinabarger§, Aoife Boyd, and Ananda


From the Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60612

Phosphomannose isomerase-guanosine 5‘-diphospho- cous exopolysaccharide called alginate (2). Alginate secreted by D-mannose pyrophosphorylase (PMI-GMP), whichis en- mucoid I! aeruginosa is a linear copolymer composed of @-1,4coded by the algA gene, catalyzes two noncontiguous linked D-mannuronic acid (partially 0-acetylated) and theC-5 steps in the alginate biosynthetic pathwayof Pseudomo- epimer L-guluronic acid (3-5). Alginate presents a substantial nas aeruginosa: the isomerization of D-fructose 6-phos- mechanical obstruction in theCF-affected lung and appears to phate to D-mannose 6-phosphate and the synthesis of reduce both the efficiency of the cellular immune responsetoGDP-D-mannose and PPifrom GTP and D-mannose ward and theeffectiveness of antibiotic therapy against €? ae1-phosphate.Amino acids that arerequired for the GMP ruginosa thatinfect the lungsof CF patients (1).Elimination of enzyme activity were identified through site-directed the alginate barrierwould likely enhance treatment of chronic mutagenesis of the algA gene. Mutation of Lys-176 to arginine, glutamine, or glutamate produced an enzyme pulmonary infections by €? aeruginosa and improve the progwhose K , for D-mannose 1-phosphatewas 4704,200-fold nosis for CF patients. Thus,we are studying thebiochemistry greater than thatmeasured forthe wild type enzyme. In and genetics of the alginate pathway in I! aeruginosa in an , for the effort to identify nontoxic inhibitors of alginate biosynthetic addition, these mutant enzymes hada lower V GMP activity as compared withthe wild type PMI-GMP. enzymes. Mucoid strains of €? aeruginosa produce low levels of the These results indicate that Lys-175is primarily involved in the binding of the substrate D-mannose 1-phosphate, alginate biosynthetic enzymes (Fig. l ) , whereas these enzymes or greatly reduced in nonmucoid strains (6,7). although it is likely that other residues are required for are either absent the specificity of binding. Mutation of k g 1 9 to gluta- Many of the alginatebiosynthetic genes have been cloned and sequenced (8-15). In addition, cloning alginate genes under mine, histidine, or leucine resulted in a 2-fold lowerV , for the GMP enzyme activity and a 4-7-fold increase in control of a strong promoter (e.g. tac) hasbeen used to produce the K , for GTP as compared withthe wild type enzyme. sufficient amounts of their respective gene productsfor identiThus, it appears that kg-19 functions in the binding of fication of enzyme function(9, 11-14, 16, 17). This strategy also GTP. In addition, chymotryptic digestion of PMI-GMP facilitated the purification of PMI-GMP (161, a bifunctional showed that the carboxyl terminus is critical for PMI enzyme that catalyzes the first and third steps of alginate activity but not for GMP activity. Taken together,these biosynthesis, and GDP-D-mannose dehydrogenase (171, which results support the hypothesis that the bifunctional catalyzes the fourth stepo f the alginate pathwayof I! aerugiPMI-GMP protein is composed of two independent en- nosa (Fig. 1). zymatic domains. The purified PMI-GMP protein exhibitsa number of characteristics which suggest that the PMI and GMP enzymatic activities may reside in separate catalytic domains (16). First, Pseudomonas aeruginosa causes severe and debilitating pul- PMI activity (but not GMP activity) is inhibited by sulfhydryl monary infections of children and young adults amicted by CF’ reagents. Second, GMP activityis dependenton the presence of (1).The abnormal exocrine mucous secretions present in the either Mg2+or Mn2+,whereas the PMI reaction utilizes a valungs of these patients induce r! aeruginosa to produce a vis- riety of divalent metals with Co2+ giving maximal activity. Third, the substrates and products of the GMP reaction do not * This work was supported in part by National Institutes of Health inhibit the PMI enzyme activity and vice uersa. Fourth, there of are large structuraldifferences betweenthe substratesfor PMI Grant AI-16790-13 and a grant from the EliLillyCo.Thecosts publication of this article were defrayedin part by the payment of page and GMP as well as mechanistic differences between an isomcharges. This article must therefore be hereby marked “uduertisement” erization reaction and the chargingof sugar phosphate with a in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. nucleotide. In this study, we used site-directed mutagenesis $ Present address: Abbott Laboratories, Hospital Products Division and limitedproteolysis to identify regions of the aZgA-encoded D-97/AP4A, 1Abbott Park Rd., Abbott Park, IL 60064. §Supported by the Cystic Fibrosis Foundation. Present address: Proc-protein necessary for the PMI and GMP enzyme activities. ter and Gamble Pharmaceuticals, Woods Corner, Route 320, Box 191, Norwich, NY 13815. EXPERIMENTALPROCEDURES 7l To whom correspondence and reprint requests should be addressed: Materials-SDS, acrylamide, bisacrylamide, Coomassie Blue G-250, Dept. of Microbiology and Immunology (WC ?go), University of Illinois TSKphenyl-5-PW College of Medicine, 901 S. Wolcott Ave., Chicago, IL 60612. Tel.: 312- Bradfordproteinassay reagent,andtheBio-Gel HPLC column were from Bio-Rad. All other chemicals were obtained 996-4586; Fax: 312-996-6415. from Sigma unless indicated otherwise. Phosphoglucose isomerase (EC The abbreviations used are CF, cystic fibrosis; PMI, phosphoman5.3.1.91, glucose-6-phosphate dehydrogenase(EC, and hexokinose isomerase; GMP, GDP-D-mannose pyrophosphorylase; HPLC, high nase (EC were also purchased from Sigma. The Q-Sepharose performance liquid chromatography; IPTG, isopropyl 1-thio-p-D-galactopryanoside; PAGE, polyacrylamide gel electrophoresis; MOPS, 4-mor-resin and Mono-Qcolumnwere from Pharmacia LKB Biotechnology, Inc. pholinepropanesulfonic acid.




Proteolysis Studies-Purified PMI-GMP (5 mg) was incubated with 250 pg of chymotrypsin for 45 min at 25 "C in the presence of 1 m~ D-mannose 1-phosphate, 1 m~ GTP, and 10 mM MgCl,. The GMP substrates were found to increase greatly the stability of the major chymotryptic fragment. The chymotrypsin reaction was then treated 250 with pg of trypsidchymotrypsin inhibitor and subsequently diluted with 2 volumes of 100 n" Tris-HC1, pH 7.0, containing 10 m~ MgCl,, 2 mM dithiothreitol, and15% glycerol. The sample was loaded ontoMono-& a column (Pharmacia) that had been equilibrated in the same buffer. Protein was eluted with 60-ml a linear gradient of C 2 5 0 m~ NaCl a t a flow rate of 1 ml/min. The column fractions containing the 52-kDa FIG.1. Alginate biosynthetic pathway of P. aeruginosa. The chymotryptic fragment werepooled, concentrated using a Centricon-10 dashed arrow indicates the steps of polymerization, acetylation, export, filter (Amicon), and then digestedsecond a time with chymotrypsin. The and epimerization. The gene encoding each enzyme is indicated. Equi- purified chymotryptic fragment was then assayed for PMI and GMP libria for the alginate reactions are indicated by the relative sizesof the activity as described above. The amino-terminal amino acid sequence arrows for each direction of the PMI, phosphomannomutase (PMM), was determined by Dr. Ka-Leung Ngai at the University of Illinois at GMP, and GDP-0-mannose dehydrogenase (GMD) reactions (6,16,17). Urbana. F6P, D-fructose 6-phosphate; M6P, D-mannose Sphosphate, MlP, Dmannose l-phosphate; GDPM, GDP-D-mannose; GDPMA, GDP-D-mannuronic acid. RESULTS AND DISCUSSION

Amino Acid Sequence Comparisons of PMI-GMP with ReStrains and Plasmids-The bacterial strains and plasmids used in this study are shown in Table I. I? aeruginosa 8821 is a n alginate- lated Proteins-The amino acid sequence of the algA-encoded producing (Alg') strain isolated from the sputumof a CF patient and, PMI-GMP was first compared with the sequences of proteins like other mucoid I? aeruginosa strains, spontaneously reverts to the that catalyze similar enzymatic reactions to identify residues nonmucoid (Alg-) form (18). The stable Alg' strain 8830 was obtained by that arepotentially important for PMI and/or GMP activity. No chemical mutagenesis of the spontaneous nonmucoid strain 8822. The algA mutant strain 8853 was isolated aRer further mutagenesis of significant areas of homology were identifiedin a comparison of strain 8830 (18). The plasmid pDSl (Fig. 21, which was used for site- PMI-GMP with the PMI enzymes of Escherichia coli (261, Salmonella typhirnurium (271, or Rhizobium meliloti (28). PMIdirected mutagenesis of the algA gene, was constructed by cloning a 2-kilobase BamHI fragment, containing the algA gene, from PES119 GMP was, however, found to sharea high degree of amino acid (12) intopUC119 using standard methods (19). sequence similarity with theGMP enzymes RfbM and CpsB of Site-directed Mutagenesis ofalgA-Mutations in thealgA gene were S. typhimurium andE. coli (Fig. 3; 29-32). The XanB protein of obtained using the oligonucleotide-directed in vitro mutagenesis system of Amersham (version 2) except that pUC119 was used as the cloning Xunthamonascampestris (331, abifunctional PMI-GMP enzyme involved in xanthan gum synthesis, was also found to be vector. Single-stranded DNA of the pUC119 aZgA derivative, pDS1, was obtained using the helper phage M13K07 (Promega). Mutant oligo- highly related to PMI-GMP of the alginate biosynthetic pathnucleotide primers were prepared by Operon Technology, Inc. (Alameda, way (Fig. 3). CA). Mutations in the algA gene were confirmed by DNA sequencing A comparison of PMI-GMP with other isomerases and pyrousing the dideoxynucleotide chain termination method (20) with the phosphorylases revealed a weak homology (

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