Penicillin V acylase crystal structure reveals new Ntn-hydrolase family members

TLDR: The crystal structure of PVA was determined to establish the nature of its catalytic mechanism and to identify any biochemical and structural relationships with PGA and other Ntn (N-terminal nucleophile) hydrolases.

Abstract: 414 nature structural biology ¥ volume 6 number 5 ¥ may 1999 Two enzyme types, penicillin V acylases (PVA) and penicillin G acylases (PGA), with distinct substrate preferences, account for all the enzymic industrial production of 6-aminopenicillanic acid 1,2. This b-lactam compound is then elaborated into a range of semi-synthetic penicillins. Although their industrial substrates are very similar, representative examples of the two enzyme types differ widely in molecular properties. PVA from Bacillus sphaericus is tetrameric with a monomer M r of 35,000 while PGA from Escherichia coli is a heterodimer of M r 90,000. Furthermore, they have no detectable sequence homology. These differences, which exist in spite of the similarity of their industrial substrates, provoked us to determine the crystal structure of PVA to establish the nature of its catalytic mechanism and to identify any biochemical and structural relationships with PGA and other Ntn (N-terminal nucleophile) hydrolases. The PVA molecule is a well-defined tetramer with 222 organization made up of two obvious dimers (A and D) and (B and C), which generate a flat disc-like assembly (Fig. 1a). The X-ray analysis revealed that the PVA monomer contains two central anti-parallel b-sheets above and below which is a pair of anti-parallel helices (Fig. 1b). There are two extensions , one from the upper pair of helices and the other at the C-terminal segment, that interact with other monomers in the tetramer and help stabilize it. The b-sheet and helix organization and connectivity are characteristic of members of the Ntn hydrolase family, which have an N-terminal catalytic residue that is often created by autocatalytic processing 3,4. In the PVA structure, cysteine was observed as the N-terminal residue, whereas the gene sequence predicts an N-terminal sequence of Met-Leu-Gly-Cys 5. This finding shows that three amino acids are processed from the precursor N-terminus to unmask a nucleophile with a free a-amino group. Since PVA is an Ntn hydro-lase, we can deduce that the N-terminal cysteine in PVA is the catalytic residue. The PVA and PGA enzymes thus share a distinctive structural core but are otherwise unrelated in primary sequence, including the active site residue. Both PGA and PVA have approximately the same angle (+30°) between the b-strands of the two b-sheets, which are decorated by the active site residues in Ntn hydro-lases. Using these b-sheets for structural alignment reveals that the catalytic regions of PVA and PGA overlap (Fig. 1c) with a root …