A secreted aminopeptidase of Pseudomonas aeruginosa -: Identification, primary structure, and relationship to other aminopeptidases

被引:59
作者
Cahan, R
Axelrad, I
Safrin, M
Ohman, DE
Kessler, E [1 ]
机构
[1] Tel Aviv Univ, Sackler Fac Med, Sheba Med Ctr, Maurice & Gabriela Goldschleger Eye Res Inst, IL-52621 Tel Hashomer, Israel
[2] Virginia Commonwealth Univ, Med Coll Virginia, Richmond, VA 23298 USA
[3] McGuire Dept Vet Affairs Med Ctr, Richmond, VA 23249 USA
关键词
D O I
10.1074/jbc.M106950200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Using leucine-p-nitroanilide (Leu-pNA) as a substrate, we demonstrated aminopeptidase activity in the culture filtrates of several Pseudomonas aeruginosa strains. The aminopeptidase was partially purified by DEAE-cellulose chromatography and found to be heat stable. The apparent molecular mass of the enzyme was similar to 56 kDa; hence, it was designated AP(56). Heating (70 degreesC) of the partially purified aminopeptidase preparations led to the conversion of AP56 to a similar to 28-kDa protein (AP(28)) that retained enzyme activity, a reaction that depended on elastase (LasB). The pH optimum for Leu-pNA hydrolysis by AP(28) was 8.5. This activity was inhibited by Zn chelators but not by inhibitors of serine- or thiol-proteases, suggesting that AP(28) is a Zn-dependent enzyme. Of several amino acid p-nitroanilide derivatives examined, Leu-pNA was the preferred substrate. The sequences of the first 20 residues of AP(56), and AP(28) were determined. A search of the P. aeruginosa genomic data base revealed a perfect match of these sequences with positions 39-58 and 273-291, respectively, in a 536-amino acid residue open reading frame predicted to encode an aminopeptidase. A search for sequence similarities with other proteins revealed 52% identity with Streptomyces griseus aminopeptidase, similar to 35% identity with Saccharomyces cerevisiae aminopeptidase Y and a hypothetical aminopeptidase from Bacillus subtilis, and 29-32% with Aeromonas caviae, Vibrio proteolyticus, and Vibrio cholerae aminopeptidases. The residues potentially involved in zinc coordination were conserved in all these proteins. Thus, P. aeruginosa aminopeptidase may belong to the same family (M28) of metalloproteases.
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页码:43645 / 43652
页数:8
相关论文
共 57 条
[1]  
AWAD WM, 1998, HDB PROTEOLYTIC ENZY, P1431
[2]   SPECIFICITY OF STREPTOMYCES-GRISEUS AMINOPEPTIDASE AND MODULATION OF ACTIVITY BY DIVALENT METAL-ION BINDING AND SUBSTITUTION [J].
BENMEIR, D ;
SPUNGIN, A ;
ASHKENAZI, R ;
BLUMBERG, S .
EUROPEAN JOURNAL OF BIOCHEMISTRY, 1993, 212 (01) :107-112
[3]   A RAPID, SENSITIVE METHOD FOR DETECTION OF ALKALINE-PHOSPHATASE CONJUGATED ANTI-ANTIBODY ON WESTERN BLOTS [J].
BLAKE, MS ;
JOHNSTON, KH ;
RUSSELLJONES, GJ ;
GOTSCHLICH, EC .
ANALYTICAL BIOCHEMISTRY, 1984, 136 (01) :175-179
[4]  
BRADFORD MM, 1976, ANAL BIOCHEM, V72, P248, DOI 10.1016/0003-2697(76)90527-3
[5]   Secretion of elastinolytic enzymes and their propeptides by Pseudomonas aeruginosa [J].
Braun, P ;
de Groot, A ;
Bitter, W ;
Tommassen, J .
JOURNAL OF BACTERIOLOGY, 1998, 180 (13) :3467-3469
[6]   Pseudomonas aeruginosa protease IV enzyme assays and comparison to other pseudomonas proteases [J].
Caballero, AR ;
Moreau, JM ;
Engel, LS ;
Marquart, ME ;
Hill, JM ;
O'Callaghan, RJ .
ANALYTICAL BIOCHEMISTRY, 2001, 290 (02) :330-337
[7]   CRYSTAL-STRUCTURE OF AEROMONAS-PROTEOLYTICA AMINOPEPTIDASE - A PROTOTYPICAL MEMBER OF THE CO-CATALYTIC ZINC ENZYME FAMILY [J].
CHEVRIER, B ;
SCHALK, C ;
DORCHYMONT, H ;
RONDEAU, JM ;
TARNUS, C ;
MORAS, D .
STRUCTURE, 1994, 2 (04) :283-291
[8]  
CHEVRIER B, 1998, HDB PROTEOLYTIC ENZY, P1433
[9]  
ELLIOTT BW, 1986, J BIOL CHEM, V261, P1259
[10]   Protease IV, a unique extracellular protease and virulence factor from Pseudomonas aeruginosa [J].
Engel, LS ;
Hill, JM ;
Caballero, AR ;
Green, LC ;
O'Callaghan, RJ .
JOURNAL OF BIOLOGICAL CHEMISTRY, 1998, 273 (27) :16792-16797