Aminopeptidases (APs) catalyze the sequential removal of amino acids from the unblocked N-termini of protein or peptide substrates, a process necessary for intracellular metabolism  and implicated in several human diseases . Most APs are metalloproteases and are classified based on substrate preference towards an acidic, basic or neutral amino acid at the P1 position of the scissile peptide bond. Very few acidic APs are known to date, the most extensively studied being the membrane-bound glutamyl aminopeptidase (ENPEP, also known as aminopeptidase A; EC 220.127.116.11) . ENPEP, a membrane-bound Ca2+-activated enzyme, is involved in the renin-angiotensin system (RAS) by catalysing the conversion of angiotensin II to angiotensin III, a key regulator of blood pressure [4, 5]. A second, cytosolic acidic AP has been reported in yeast, fungi and mammals, and termed aspartyl aminopeptidase (DNPEP, also known as DAP; EC 18.104.22.168) due to its preference for aspartate over glutamate at the P1 position [6–8]. In mammals, DNPEP is preferentially expressed and has high enzymatic activity in neurons and neuroendocrine tissues [6, 9, 10]. Its reported conversion of angiotensin I to angiotensin 2–10 , and of angiotensin II to angiotensin III in vitro, implicates a role in RAS and regulation of blood pressure. Moreover, a mild antagonist effect of DNPEP towards the bone morphogenetic protein signalling pathway has recently been reported .
DNPEP is the sole mammalian entry for the M18 metallopeptidase family, which contains ~600 putative members from bacteria and eukaryotes . The M18 family, together with the M20, M28 and M42 families, are classified into the metalloprotease H (MH) clan of proteases on the basis of active site sequence conservation according to the MEROPS database [12, 13]. Only a handful of M18 enzymes have been biochemically characterized in any detail; these include yeast vacuole aminopeptidase I (API, also known as Lap4) with a broad substrate specificity for non-polar amino acids , as well as yeast yhr113w (also known as Ape4)  and mammalian DNPEP which prefer an acidic amino acid. These M18 enzymes are shown to homo-oligomerize, reminiscent of the self-compartmentalization strategy in the well-characterized proteasomes to confer specificity towards unfolded polypeptides and not folded proteins . However, the reported dodecameric form in yeast Lap4 and Ape4 [7, 14] contrasts with the proposed octameric form in DNPEP .
Little is known about the structure-function relationship of DNPEP and other M18 members, which contain a binuclear metal centre in the active site but lack the signature Zn2+-binding sequence motif (HExxH + E) found in other metalloproteases such as ENPEP . Although several conserved histidines essential for catalysis have been identified in human DNPEP , their roles are yet to be elucidated. In this study we determined the crystal structure of human DNPEP (hDNPEP) complexed with catalytic Zn2+ and substrate analogue L-aspartate-β-hydroxamate (ABH), and confirmed its dodecameric architecture by electron microscopy (EM). The bound ABH ligand highlights the importance of a domain-swapped loop in constructing the active site and provides a structural basis for hDNPEP’s catalytic mechanism and substrate specificity. By comparison with available bacterial M18 structures we further develop a family-wide description of this unannotated peptidase family and suggest unifying catalytic features across the MH clan.