Structurally Linked Dynamics in Lactate Dehydrogenases of Evolutionarily Distinct Species

Abstract

We present new findings about how primary and secondary structure affects the role of fast protein motions in the reaction coordinates of enzymatic reactions. Using transition path sampling and committor distribution analysis, we examined the difference in the role of these fast protein motions in the reaction coordinate of lactate dehydrogenases (LDHs) of Apicomplexa organisms Plasmodium falciparum and Cryptosporidium parvum. Having evolved separately from a common malate dehydrogenase ancestor, the two enzymes exhibit several important structural differences, notably a five-amino acid insertion in the active site loop of P. falciparum LDH. We find that these active site differences between the two organisms' LDHs likely cause a decrease in the contribution of the previously determined LDH rate-promoting vibration to the reaction coordinate of P. falciparum LDH compared to that of C. parvum LDH, specifically in the coupling of the rate-promoting vibration and the hydride transfer. This effect, while subtle, directly shows how changes in structure near the active site of LDH alter catalytically important motions. Insights provided by studying these alterations would prove to be useful in identifying LDH inhibitors that specifically target the isozymes of these parasitic organisms.

Figure 1

Depiction of structural features in pfLDH (red) and cpLDH (blue). (a) Alignment of pfLDH and cpLDH crystal structures, showing the NADH (purple), substrate (dark blue), and loop insertion in pfLDH (yellow). Alignment performed with THESEUS.^, (b) Active site of pfLDH (and representative of cpLDH). Important residues are labeled. (c) Active site of pfLDH, showing the QM region (dark blue), analogous RPV residues from hhLDH (red), and L163 (yellow). (d) Active site of pfLDH, showing the QM region (dark blue), analogous RPV residues from hhLDH (red), and M163 (yellow).

Figure 2

Representative trajectories of pfLDH and cpLDH. Note the proton donor–acceptor (red), hydride donor–acceptor (black), hydride–donor and –acceptor (solid blue and dashed blue), and proton–donor and –acceptor (solid green and dashed green) distances.

Figure 3

Committor distributions of pfLDH (left) and cpLDH (right), with constraints on hydride and proton distances and the hydride dihedral (a and b), the quantum region (c and d), and the quantum region with residues analogous to the hhLDH RPV (I32, G33, G34, and R109) (e and f).

Figure 4

Representative trajectories from pfLDH and cpLDH, showing the hydride transfer (donor in red, acceptor in black), hydride donor–acceptor (green), I32–NC4 distance (blue), and transition state, as determined by committor analysis (dashed black). These distances show RPV compression relaxation occurring at different times relative to hydride transfer in the two systems.

Figure 5

Three-dimensional histograms of all structures created along all reactive trajectories from pfLDH and cpLDH projected onto the plane of the I32–NC4, the closest RPV residue and the hydride donor, distance vs the hydride transfer (the difference of the hydride bond breaking and bond forming distances). In the heat map, red indicates relatively higher probability and white indicates relatively lower probability. Contour relief lines designate lines of equal probability. It is important to note that, as these are histograms, the probability of the two histograms cannot be compared to one another and that the relatively long I32–NC4 distance (4.0–5.0 Å) is due to the distance being measured from the I32 C_δ atom, not the C_δ hydrogens.