Figure 1.
Phylogenetic reconstruction based on orthologous glycerol kinase protein sequences.
The unrooted radial tree was generated using the maximum parsimony method. A maximum likelihood tree demonstrated the same topology. Bootstrapping provided >50% support for each node. The major branches comprising the protozoa, metazoa, apicomplexa and vertebrates were analyzed to identify differences in the patterns of sequence evolution between the four groups.
Figure 2.
Posterior probabilities for four categories of ω across GK coding sequences.
The Bayes Empirical Bayes posterior probability estimates (BEB) determined from the PAML analysis of the protozoan and metazoan clades were plotted for each category of ω across the P. falciparum (PfGK) and human (HsGK) GK coding sequences, respectively. Residues under extreme purifying selection (blue lines) are potential drug target sites. Categories for ω are indicated with colored bars: yellow (ω>1.0, positive selection), red (ω = 1.0, neutral selection), white (0.1<ω<1.0, purifying selection), and blue (ω≤0.1, extreme purifying selection). The ω<0.1 category was defined by combining PAML (BEB>99%) and SLR (p<0.01) data.
Table 1.
Parameter estimates, log-likelihood values and selective pressures across codons for P. falciparum glycerol kinase under models of variable ω ratios.
Figure 3.
Multiple sequence alignment of PfGK, HsGK and EcGK.
To identify differences in selective constraints, PfGK and HsGK were analyzed separately, as part of protozoan and metazoan clades, respectively, and compared. Residues under extreme purifying selection are highlighted in yellow in PfGK and are in brown font in HsGK. Residues highlighted in gray in PfGK are under neutral or positive selection. Sites that bind glycerol (green circle), ADP (blue star) and magnesium (red triangle) were determined from the crystal structure of EcGK [24]. Corresponding residues in PfGK were determined form the MSA. Residues are numbered according to the PfGK sequence. Potential drug target sites are indicated (red bar) and labeled with Roman numerals. Underlined regions in EcGK are implicated as follows: intersubunit interactions (black), FBP regulatory site (green) and IIAGlc regulatory site (orange).
Table 2.
Summary of the location and function of potential PfGK drug target sites.
Figure 4.
Models A (EcGK) and B (PfGK) are displayed as ribbon structures with functional residues illustrated as sticks. (A) An EcGK monomer with residues involved in binding to ADP (blue), Mg2+ (yellow), glycerol (red), FBP (orange) and IIAGLC (purple) [24], [70]. Positions of Domains I and II are labeled. α Helices and ß sheets within Domains I and II form the ATPase domain, shaded in light green and areas involved in subunit interactions are shaded in aquamarine. (B) The PfGK model showing functional residues based on EcGK. (C) PfGK ribbon structure and (D) surface model showing potential drug target sites. Residues within each region are summarized in Table 2 and are highlighted as follows: Region I (magenta), Region II (red), Region III (black), Region IV (blue), Region V (orange) and Region VI (green). Region III is below Region I and is difficult to visualize in the ribbon configuration (C) since it is partly obscured by one of the helices. It is within the core of the molecule and cannot be seen on the surface PfGK model (D).
Figure 5.
rPfGK purification and enzyme activity.
(A) SDS PAGE analysis and (B) immunoblot analysis showing the expression and affinity purification of rPfGK from the soluble protein fraction of E. coli cells. Lane 1: total E. coli protein, Lane 2: soluble protein fraction, Lane 3: insoluble protein fraction, Lane 4: purified rPfGK, Lane 5: red cell membrane marker. (C) The oxidation of NADH to NAD+ in the glycerol kinase coupled enzyme reaction measured at 340nm shows active rPfGK (green) compared to the blank (black), negative control GST (red) and the positive control EcGK (blue).