Crystal Structure of Vat(D):  An Acetyltransferase That Inactivates Streptogramin Group A Antibiotics^,

Abstract

The streptogramin class of antibiotics act to inhibit bacterial protein synthesis, and their semisynthetic derivatives, such as dalfopristin-quinupristin (Synercid), are used to treat serious or life-threatening infections due to multiply antibiotic resistant bacteria. Acquired resistance of the nosocomial pathogen Enterococcus faecium to the group A component of natural and semisynthetic streptogramin mixtures is a prerequisite for the streptogramin resistance phenotype and is mediated by a streptogramin acetyltransferase. The crystal structure of Vat(D), a streptogramin acetyltransferase from a human urinary isolate of E. faecium, has been determined as an apoenzyme and in complex with either acetyl-CoA or virginiamycin M1 and CoA. These structures illustrate the location and arrangement of residues at the active site, and point to His 82 as a residue that may function as a general base. The structural similarity of Vat(D) to the xenobiotic acetyltransferase from Pseudomonas aeruginosa indicates similarities in the catalytic mechanism for these enzymes as well as several shared and distinctive antibiotic binding interactions between these enzymes and their respective substrates. These results reveal the molecular basis for a reaction by which Gram-positive cocci acquire resistance to a last resort antibiotic.

Crystal Structure and Catalytic Mechanism of PglD from Campylobacter jejuni

“…The finding that His-125 serves as the key residue for catalysis was consistent with the role played by key histidines in other L␤H enzymes (38,40,41,45). Another notable theme that recurs in L␤H enzymes is the existence of a negative dipole interacting with the key catalytic histidine (38,41,43,47). This component of the dyad serves to increase the basicity of the histidine, thus enhancing the ability to act as a general base by abstracting a proton from the substrate to be acylated.…”

Structural Basis of Synercid® (Quinupristin-Dalfopristin) Resistance in Gram-positive Bacterial Pathogens

“…common with other CoA-dependent acyltransferases containing an L␤H domain (33)(34)(35) and as demonstrated previously for VatD (11), both substrates are bound at the interfaces between subunits. The structure of the VatD apoenzyme reveals a short tunnel formed, at the N-terminal end, by residues of the extended loop region of one monomer and the L␤H domain of the symmetry-related subunit and, at the C-terminal end, by residues provided by two adjacent L␤H domains.…”

“…The crystal structure of VatD from E. faecium strain BM4145 (6) was determined recently at 2.5 Å resolution in the absence of ligands and in complex with substrates AcCoA and virginiamycin M1 (VM) at 2.7 and 2.8 Å resolution, respectively (11). In this report, we extend the structural analysis of the apoenzyme to 1.8 Å resolution and present 2.8 Å data for a complex of VatD and dalfopristin.…”

“…Atoms, other than S (yellow) and the carbon atoms of dalfopristin (green), are colored as in a, and secondary structural elements of VatD are colored in red, green, and yellow for helices, ␤-strands, and turns, respectively. d, stereo image showing the conformation of dalfopristin (green) bound to VatD superimposed on the complex of the same enzyme with VM (pink) (11). Non-carbon atoms are colored as in c. The altered positions of the pyrollidine groups (and proximal elements of the macrocycle) arise because the C-1-C-2 double bond of VM becomes saturated, when the additional C-2 substituent is introduced to produce dalfopristin, changing the pyrollidine ring from a planar to a puckered conformation.…”

Structural basis for streptogramin B resistance in Staphylococcus aureus by virginiamycin B lyase

“…However, only two clinically relevant mechanisms of antibiotic inactivation have thus far been identified. Type A streptogramins are modified by a cadre of Vat enzymes that catalyze acetylation of the antibiotic (9,10), and type B streptogramins are inactivated by a ringopening lyase called virginiamycin B lyase (Vgb) (11).…”