Crystal structure of acetoacetyl-CoA reductase from Rickettsia felis

Abstract

Rickettsia felis, a Gram-negative bacterium that causes spotted fever, is of increasing interest as an emerging human pathogen. R. felis and several other Rickettsia strains are classed as National Institute of Allergy and Infectious Diseases priority pathogens. In recent years, R. felis has been shown to be adaptable to a wide range of hosts, and many fevers of unknown origin are now being attributed to this infectious agent. Here, the structure of acetoacetyl-CoA reductase from R. felis is reported at a resolution of 2.0 Å. While R. felis acetoacetyl-CoA reductase shares less than 50% sequence identity with its closest homologs, it adopts a fold common to other short-chain dehydrogenase/reductase (SDR) family members, such as the fatty-acid synthesis II enzyme FabG from the prominent pathogens Staphylococcus aureus and Bacillus anthracis. Continued characterization of the Rickettsia proteome may prove to be an effective means of finding new avenues of treatment through comparative structural studies.

Keywords: PhaB; Rickettsia; SSGCID; Seattle Structural Genomics Center for Infectious Disease; acetoacetyl-CoA reductase; oxidoreductase; structural genomics.

Figure 1

SDS–PAGE and SEC analyses of purified recombinant R. felis acetoacetyl-CoA reductase. (a) Acetoacetyl-CoA reductase eluted as a single peak (blue line) from a HiLoad 16/60 Superdex 200 pg SEC column. The SEC column was calibrated separately (gray line), and the molecular weights of calibration protein standards from a kit are indicated. The elution volume of R. felis acetoacetyl-CoA reductase falls between those of ovalbumin (75 kDa) and aldolase (158 kDa), suggesting a higher oligomeric state. SEC-purified acetoacetyl-CoA reductase was resolved on 12% SDS–PAGE visualized with Coomassie Blue (inset); the monomer runs as a single band (AcAc-CoA reductase) near the 25 kDa protein standard marker (lane M, labeled in kDa). A standard curve for SDS–PAGE (b) and a SEC calibration curve (c) were generated using the known protein standards. In (b), plotting the molecular weights (MW) of the protein standards versus their observed R _f (relative migration; black circles), the MW of the protein monomer was estimated to be 26 kDa (blue circle). In (c), the calibration curve was obtained by plotting K _av (the partition coefficient calculated using individual elution volumes) versus log relative molecular weight (M _r) of the protein standards. The M _r of R. felis acetoacetyl-CoA reductase was determined to be approximately 101 kDa (blue circle), indicating a tetrameric form, which is common in the short-chain dehydrogenase/reductase (SDR) family.

Figure 2

Crystal structure of R. felis acetoacetyl-CoA reductase. (a) Ribbon diagram of the R. felis acetoacetyl-CoA reductase tetramer. Helices are colored in magenta and strands in green, with a representative monomer highlighted in violet. Acetoacetyl-CoA reductase crystallized with two molecules per asymmetric unit, and a tetramer was generated by crystallographic symmetry. (b) Representative monomer of R. felis acetoacetyl-CoA reductase with secondary-structure elements labeled. The active-site residues (Ser135, Tyr148 and Lys152) are grouped in helix α6 and the connecting loop to β5.

Figure 3

Primary-sequence alignment of acetoacetyl-CoA reductases from R. felis (PDB entry 4kms) and B. pseudomallei (PDB entry 3ezl) and of FabGs from S. aureus (PDB entry 3osu) and B. anthracis (PDB entry 2uvd). Secondary-structural elements shown include α-helices (α), 3₁₀-helices (η), β-strands (β) and β-turns (TT). Identical residues are shown in white on a red background, while conserved residues are shown in red and related residues are outlined in blue. Asterisks indicate conserved residues implicated in functional specificity. The image was generated using ESPript (Robert & Gouet, 2014 ▸).

Figure 4

Active site of R. felis acetoacetyl-CoA reductase. The catalytic triad of R. felis (Ser135, Tyr148 and Lys152) found in helix α6 and the connecting loop to β5 is shown as sticks (magenta, top panel). Alignment of R. felis acetoacetyl-CoA reductase with NADP⁺-bound B. pseudomallei aceto­acetyl-CoA reductase (green; catalytic triad shown as sticks) demonstrates necessary rearrangements to the active site, including movement of Ser135 and reorientation of Lys152, to accommodate substrates (lower panel).