Puromycin, a selective inhibitor of PSA acts as a substrate for other M1 family aminopeptidases: Biochemical and structural basis Ravikumar Reddi a,b,1 , Roopa Jones Ganji a,1 , Anil Kumar Marapaka a,b,1 , Sandeep Chowdary Bala a,b , Naga Veera Yerra b,c , Neshatul Haque a , Anthony Addlagatta a,b, ⁎ a Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Telangana, India b Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India c Department of Analytical and Structural Chemistry, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, Telangana, India abstract article info Article history: Received 6 April 2020 Received in revised form 1 October 2020 Accepted 5 October 2020 Available online 09 October 2020 Keywords: M1 class aminopeptidases Human puromycin sensitive aminopeptidase PSA Selective inhibitor Aminopeptidase N Puromycin O-methyl-L-tyrosine Puromycin sensitive aminopeptidase (PSA or NPEPPS) is a M1 class aminopeptidase is selectively inhibited by the natural product puromycin, an aminonucleoside antibiotic produced by the bacterium Streptomyces alboniger. The molecular basis for this selective inhibition has not been understood well. Here, we report the basis for selectivity of puromycin using biochemical, structural and molecular modeling tools on four different M1 family enzymes in- cluding human PSA. Except for PSA, the other three enzymes were not inhibited. Instead, the peptide bond in the puromycin is hydrolyzed to O-methyl-L-tyrosine (OMT) and puromycin aminonucleoside (PAN). Neither of the hy- drolyzed products, individually or together inhibit any of the four enzymes. Crystal structure of ePepN using crystals that are incubated with puromycin contained the hydrolyzed products instead of intact puromycin. On the other hand, intact puromycin molecule was observed in the crystal structure of the inactive mutant ePepN (E298A)-puro- mycin complex. Surprisingly, puromycin does not enter the active site of the mutant enzyme but binds near the en- trance. Comparison of puromycin binding region in ePepN mutant enzyme and molecular modeling studies suggest that PSA might be inhibited by similar mode of binding there by blocking the entrance of the active site. © 2020 Elsevier B.V. All rights reserved. 1. Introduction Puromycin is a natural product produced by Streptomyces alboniger and is a nucleoside with O-methyl-L-tyrosine (OMT) conjugated to pu- romycin aminonucleoside (PAN) by a peptide bond (Fig. 1)[1]. Puromy- cin is a well-known protein synthesis inhibitor and works by disrupting peptide transfer on ribosomes causing premature chain termination during translation [2]. Apart from protein synthesis inhibition, puromy- cin is also implicated in the selective inhibition of puromycin sensitive aminopeptidases (PSA) also referred as NPEPPS, a member of the M1 family aminopeptidases [4]. Humans have at least 12 different M1 class aminopeptidases that include the extracellular membrane- associated aminopeptidase N also called as CD13 (hAPN) and puromy- cin sensitive aminopeptidase (PSA) among several others. M1 class ami- nopeptidases are metalloproteases with the HEXXH-(X) 18 -E zinc- binding sequence and harbors exopeptidase motif, GXMEN, in the active site [5]. They cleave single amino acids from the amino terminus of the peptide [10]. All human enzymes of this family have been implicated to play a critical role in the normal physiology and in pathogenesis [11]. Based on the reported literature, of all the M1 class aminopeptidases, puromycin inhibits PSA very selectively [17,18]. There have been no ef- forts to understand why only PSA is inhibited and not others in the M1 family. Given the biochemical similarities, it is expected that PSA shares a similar active site with other homologous proteins. In this study, we aimed at understanding the molecular basis for selective inhibition of PSA by puromycin using biochemical and structural tools. In this study, we have used human PSA, E. coli aminopeptidase N (ePepN), Plas- modium falciparum aminopeptidase N (PfAPN) and porcine microsomal aminopeptidase N (PmAPN). Biochemical, structural and molecular modeling studies suggest that the puromycin binds at the entrance of the active site of PSA while in other members of M1 family aminopepti- dases, it enters like a peptide substrate and gets cleaved thus providing the basis for selectivity. 2. Materials and methods 2.1. Materials Puromycin dihydrochloride, puromycin aminonucleoside (PAN), O- methyl-L-tyrosine (OMT), PmAPN and Leu-pNA, nickel affinity resin International Journal of Biological Macromolecules 165 (2020) 1373–1381 ⁎ Corresponding author at: Department of Applied Biology, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, Telangana, India. E-mail address: anthony@iict.res.in (A. Addlagatta). 1 These authors made an equal contribution. https://doi.org/10.1016/j.ijbiomac.2020.10.035 0141-8130/© 2020 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac