Crystal structure and solution scattering of Geobacillus stearothermophilus S9 peptidase reveal structural adaptations for carboxypeptidase activity.

Acylaminoacyl peptidases (AAPs) play a pivotal role in various pathological conditions and are recognized as potential therapeutic targets. AAPs exhibit a wide range of activities, such as acylated amino acid-dependent aminopeptidase, endopeptidase, and less studied carboxypeptidase activity. We have determined the crystal structure of an AAP from Geobacillus stearothermophilus (S9gs) at 2.0 Å resolution. Despite being annotated as an aminopeptidase in the NCBI database, our enzymatic characterization proved S9gs to be a carboxypeptidase. Solution-scattering studies showed that S9gs exists as a tetramer in solution, and crystal structure analysis revealed adaptations responsible for the carboxypeptidase activity of S9gs. The findings present a hypothesis for substrate selection, substrate entry, and product exit from the active site, enriching our understanding of this rare carboxypeptidase.

Assessing the prospect of XAFS experiments of metalloproteins under in vivo conditions at Indus-2 synchrotron facility, India.

The feasibility of X-ray absorption fine-structure (XAFS) experiments of ultra-dilute metalloproteins under in vivo conditions (T = 300 K, pH = 7) at the BL-9 bending-magnet beamline (Indus-2) is reported, using as an example analogous synthetic Zn (0.1 mM) M1dr solution. The (Zn K-edge) XAFS of M1dr solution was measured with a four-element silicon drift detector. The first-shell fit was tested and found to be robust against statistical noise, generating reliable nearest-neighbor bond results. The results are found to be invariant between physiological and non-physiological conditions, which confirms the robust coordination chemistry of Zn with important biological implications. The scope of improving spectral quality for accommodation of higher-shell analysis is addressed.

Catalytic triad heterogeneity in S51 peptidase family: Structural basis for functional variability.

Peptidase E (PepE) is a nonclassical serine peptidase with a Ser-His-Glu catalytic triad. It is specific for dipeptides with an N-terminal aspartate residue (Asp-X dipeptidase activity). Its homolog from Listeria monocytogenes (PepElm) has a Ser-His-Asn "catalytic triad." Based on sequence alignment we predicted that the PepE homolog from Deinococcus radiodurans (PepEdr) would have a Ser-His-Asp "catalytic triad." We confirmed this by solving the crystal structure of PepEdr to 2.7 Å resolution. We show that PepElm and PepEdr lack the Asp-X dipeptidase activity. Our analyses suggest that absence of P1 pocket in the active site could be the main reason for this lack of typical activity. Sequence and structural data reveal that the PepE homologs can be divided into long and short PepEs based on presence or absence of a C-terminal tail which adopts a ß-hairpin conformation in the canonical PepE from Salmonella enterica. A long PepE from Bacillus subtilis with Ser-His-Asp catalytic triad exhibits Asp-X dipeptidase activity. Whereas the three long PepEs enzymatically characterized till date have been found to possess the Asp-X dipeptidase activity, the three enzymatically characterized short PepEs lack this activity irrespective of the nature of their catalytic triads. This study illuminates the structural and functional heterogeneity in the S51 family and also provides structural basis for the functional variability among PepE homologs.