Paenidase, a Novel d-Aspartyl Endopeptidase from Paenibacillus sp. B38: Purification and Substrate Specificity

Abstract

We discovered and characterized a novel type D-aspartyl endopeptidase (DAEP) produced extracellularly by Paenibacillus sp. B38. This bacterial DAEP of M(r) 34,798 (named paenidase) appeared to be converted into a smaller form of M(r) 34,169 by the proteolytic removal of 5 amino acid residues from the N-terminal. The intact and modified forms of the enzyme displayed essentially the same enzymatic properties. The enzyme specifically hydrolyzed succinyl-D-aspartic acid alpha-(p-nitroanilide) and succinyl-D-aspartic acid alpha-(4-methylcoumaryl-7-amide) to generate p-nitroaniline and 7-amino-4-methylcoumarin, and internally cleaved a synthetic peptide (D-A-E-F-R-H-[D-Asp]-G-S-Y) of the [D-Asp](7) amyloid beta (Abeta) protein between [D-Asp](7)-G(8). Either was totally inert to the normal Abeta peptide sequence containing L-Asp, instead of D-Asp at the 7th position. Thus, paenidase is the first DAEP from a microorganism that specifically recognizes an internal D-Asp residue to cleave [D-Asp]-X peptide bonds.

B38-CAP is a bacteria-derived ACE2-like enzyme that suppresses hypertension and cardiac dysfunction

“…Genomic DNAs were isolated from Paenibacillus sp. B38 22 Supplementary Table 4. All values are means ± SEM.…”

“…We had previously cloned a Daspartyl endopeptidase (paenidase I) from Paenibacillus sp. B38, a new substrain of B. subtilis 22,23 . Paenidase I cleaves D-α-Aspcontaining amyloid-β peptide, which is detected in Alzheimer's disease, suggesting a potential application as a therapeutic 22 .…”

Site‐specific rapid deamidation and isomerization in human lens αA‐crystallin in vitro

“…It should be noted that PIMT restores l ‐β‐Asp to the five‐membered ring l ‐succinimide intermediate but, because the rate of hydrolyzation to β‐Asp is three times as fast as the rate of hydrolyzation to α‐Asp, much of the peak would remain in the original position . Paenidase is a d ‐aspartyl endopeptidase that cleaves the peptide bond between a d ‐α‐Asp residue and its C‐terminal neighboring residue . Incubation with this enzyme did not affect any peptide peaks (Figure 3d); consequently, there were no peptides containing d ‐α‐Asp.…”

“…19 Paenidase is a D-aspartyl endopeptidase that cleaves the peptide bond between a D-α-Asp residue and its Cterminal neighboring residue. 3,20 Incubation with this enzyme did not affect any peptide peaks ( Figure 3d); consequently, there were no peptides containing D-α-Asp. The remaining small peak (D3) was not digested by any of the enzymes, indicating that this peptide contained D-β-Asp.…”

The protective role of protein L-isoaspartyl (D-aspartate)O-methyltransferase for maintenance of mitochondrial morphology in A549 cell

“…The Paenidase I enzyme; it showed little activity towards L-Asp and L-and D-isoAsp but demonstrated specific higher activity specifically for D-Asp residues and insensitivity toward protease inhibitors. [30] Therefore, we elected to use this enzyme to detect D-Asp modifications in proteins from the perspective of a comprehensive detection of the proteins with D-Asp residues, although this method has a limited ability to characterize proteins with only D-Asp residue. This method, however, enables the direct detection of D-Asp residue for effectively screening the proteins with D-Asp residues and differs from analysis methods using protein purification followed by HPLC.…”