Fig 1.
Structural dynamics in Adkeco and positions of mutations.
(A) Structure of the open and substrate free conformation of Adkeco [16] (4AKE.pdb). Positions mutated in this study are indicated with ball and stick representations and colored in gold. (B) Crystallographic structure of Adkeco in the closed and active conformation [17] (1AKE.pdb). The inhibitor Ap5A [29] is displayed with a ball and stick representation.
Fig 2.
Structures of yeast and E. coli adenylate kinase in closed and active states.
The stereo-view was made by superposition of Cα atoms of Adk1yeast [26] (2AKY) and Adkeco [17] (1AKE.pdb). The inhibitor Ap5A [29] is displayed with a ball and stick representation. Adk1yeast and the corresponding Ap5A molecule is colored blue while Adkeco with its corresponding Ap5A molecule is colored red.
Table 1.
Nomenclature, catalytic parameters and melting temperatures of Adk variants.
Fig 3.
Catalytic activity of Adk variants at 20°C.
Adk variants analyzed were designed to have a broad coverage of kcat which is illustrated by a display of kcat from Table 1 vs the corresponding Adk variant.
Fig 4.
NMR spectra of yeast Adk1 and E. coli Adk.
(A) 1H-15N HSQC spectrum of Adk1yeast (B) 1H-15N HSQC spectrum of Adkeco. The spectra were acquired at 20°C and show that both enzymes are properly folded at the conditions used in this study.
Fig 5.
Thermal stability of Adk variants.
The thermal stability of Adk1yeast (black), (red),
(green),
(blue),
(turquoise),
(pink),
(purple),
(yellow) and
(olive) was quantified by observing normalized circular dichroism signals at a wavelength of 220 nm as a function of temperature. The data are displayed assuming a two-state unfolding model. Associated melting temperatures (TM) are displayed in Table 1.
Fig 6.
Yeast plasmid shuffling assay system.
The yeast ADK1 open reading frame was exchanged with the KanMX cassette. Viability of the resulting strain depends on the presence of a wild-type yeast ADK1 gene in a low-copy number URA3-based vector, pRS316. A second low-copy number LEU2-based vector was used to introduce different alleles of the E. coli adk gene into this strain. Thus, a strain harboring both the URA3 plasmid (wild-type yeast ADK1 gene) and the LEU2 plasmid (mutated E. coli adk gene) can be obtained. If such a strain is plated on medium containing 5-FOA, the URA3 vector will be counter-selected as the URA3 gene product converts 5-FOA to a toxic compound [22]. Thus, this plasmid shuffling procedure can reveal the phenotype conferred by a mutated E. coli adk gene located in the LEU2 plasmid.
Fig 7.
Serial dilution growth assays at 30°C and 20°C.
Yeast adk1Δ cells expressing Adk1yeast or indicated variants of Adkeco proteins were serially diluted, spotted on SC-Leu plates, and incubated at 30°C and 20°C for 3–4 days.
Table 2.
Expression levels, growth rate constants, relative fitness and apparent kcat values of Adkeco variants at 20°C.
Fig 8.
Dependencies of relative fitness on Adk catalytic parameters kcat, and kcat/
.
(A) Relative fitness plotted versus kcat. (B) Relative fitness plotted versus the Michaelis constant (KM). (C) Relative fitness plotted versus the specificity constant (kcat/). Cells were cultivated for growth rate measurements at 20°C. Catalytic parameters (kcat and
) were obtained from a coupled ATPase assay [33]. Error bars for growth rate constants indicate standard deviations obtained from three independent biological replicates. Error bars for Adk catalytic parameters kcat and
indicate standard deviations of three technical replicates.
Fig 9.
Effect of Adkeco expression levels in yeast cells.
(A) Western blot analysis of Adkeco proteins expressed from the pRS315 vector in adk1Δ yeast cells and detected by a rabbit polyclonal anti-Adkeco antibody. Endogenous Act1p was used as a loading control. Protein levels of the Adkeco variants were normalized with respect to wild-type protein levels (Table 2). (B) Increases in Adkeco protein levels correlate with reductions in catalytic activity (kcat). (C) Relative fitness plotted versus
(i.e kcat normalized with respect to Adkeco protein levels according to Eq 2). Cells were cultivated at 20°C for western blots. Error bars for protein levels indicate standard deviations obtained from three independent biological replicates.