Identification of a new chromophoric substrate in the library of amino acid p-nitroanilides for continuous assay of VanX, a D,D-dipeptidase essential for vancomycin resistance.
As one of key bacterial proteins involved in vancomycin resistance, VanX is a D,D-dipeptidase that impedes bacterial cell wall biosynthesis by hydrolyzing the essential D-Ala-D-Ala dipeptide. Based on a report by Crowder and co-workers that L-alanine-p-nitroanilide (L-Ala-pNA) was a useful substrate for continuous assay of VanX, we constructed a library of 35 L- and D-amino acid p-nitroanilides to provide the needed diversity to discover new substrates that are more specific than L-Ala-pNA. We report here that, among all compounds tested, D-leucine-p-nitroanilide (D-Leu-pNA) was found to be the best substrate for VanX enzyme (KM=8.9+/-1.2 mM, kcat=0.0102+/-0.0016 s(-1), kcat/KM=0.0012 mM(-1)s(-1)). Although it is catalytically inefficient, this new VanX substrate needs essentially no sophisticated synthetic chemistry for preparation and therefore offers a convenient means for routine analysis of enzyme catalysis and the screening of potential inhibitors. Moreover, because it is the uncommon leucine in its D form in D-Leu-pNA, enzymatic activities due to other contaminated species in Escherichia coli used for VanX overproduction should be greatly reduced.
Hsieh
Ming-Lung
ML
Department of Chemistry and Biochemistry, National Chung Cheng University, Chia-Yi, Taiwan 621, Republic of China.
Tseng
Min-Jen
MJ
Tseng
Ming-Chung
MC
Chu
Yen-Ho
YH
D000813:Anilides / Q000737:chemistry*; D000814:Aniline Compounds / Q000032:analysis / Q000737:chemistry; D001426:Bacterial Proteins / Q000032:analysis*; D002384:Catalysis; D002863:Chromogenic Compounds; D004150:Dipeptidases / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D007536:Isomerism; D007700:Kinetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase / Q000032:analysis*; D013379:Substrate Specificity; D020713:Vancomycin Resistance
D000813:Anilides; D000814:Aniline Compounds; D001426:Bacterial Proteins; D002863:Chromogenic Compounds; D011993:Recombinant Fusion Proteins; C004813:alanine-4-nitroanilide; C001536:1-leucine-4-nitroanilide; D004150:Dipeptidases; D046929:Serine-Type D-Ala-D-Ala Carboxypeptidase; C091956:VanX dipeptidase