The 2.0 Å Crystal Structure of Cephalosporin Acylase

Abstract

The substrate binding pocket of the P. diminuta cephalosporin acylase provides detailed insight into the ten key residues responsible for the specificity of the cephalosporin C side chain in four classes of cephalosporin acylases, and it thereby forms a basis for the design of an enzyme with an improved conversion rate of CPC to 7-ACA. The structure also provides structural evidence that four of the five different classes of cephalosporin acylases can be grouped into one family of the Ntn hydrolase superfamily.

Precursor Structure of Cephalosporin Acylase: Insights into Auto-Proteolytic Activation in a New N-Terminal Hydrolase Family

“…1) from a class I CA from Pseudomonas diminuta (CAD) at 2.5 Å resolution [2]. A conserved water molecule, stabilized by four hydrogen bonds in unusual pseudo-tetrahedral geometry, plays a key role to assist the OG atom of Ser1β to generate a strong nucleophile (Fig.…”

“…The precursor structure is a single chain protein, consisting of the α subunit of active CAD in green, an orange colored P-loop (spacer structure in loop form), and the β-subunit of CAD in purple. The side-chain pocket for binding the substrate glutaryl-7-aminocephalosporanic acid is represented by a ball-and stick model in the center [2]. a b FIGURE 2.…”

“…(a) The conserved water of precursor CA. The OG atom of Ser1β in the S1βA precursor CA was modeled after the torsional geometry of the OG atom of Ser1β residue of CA structure [2]. (b) The conserved water of CA.…”

The N-terminal Nucleophile Serine of Cephalosporin Acylase Executes the Second Autoproteolytic Cleavage and Acylpeptide Hydrolysis

“…Ntn hydrolases feature an ␣␤␤␣ sandwich and an N-terminal, catalytically active, nucleophile that is released by the first proteolytic step (5)(6)(7). Examples of Ntn hydrolases are glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase (8), proteasome ␤-subunits (9), penicillin acylases (7), glucosamine-6-phosphate synthase (10), isoaspartyl aminopeptidase (11), taspase 1 (12), glycosylasparaginase (GA) (13,14), the human asparaginase-like protein 1 (hASRGL1) (15), Helicobacter pylori ␥-glutamyltranspeptidase (16), N-acylhomoserine lactone acylase PvdQ (PvdQ) (17), and cephalosporin acylases (18) whose crystal structures are available.…”

“…The spacer is expelled from its position in the CA, and has no further use. The mature ␣-and ␤-subunits stay together and form the mature, active, heterodimeric CA (18,26).…”