Structural Characterization and Functional Analysis of Mevalonate Kinase from Tribolium castaneum (Red Flour Beetle)

Abstract

Mevalonate kinase (MevK) is an important enzyme in the mevalonate pathway that catalyzes the phosphorylation of mevalonate into phosphomevalonate and is involved in juvenile hormone biosynthesis. Herein, we present a structure model of MevK from the red flour beetle Tribolium castaneum (TcMevK), which adopts a compact α/β conformation that can be divided into two parts: an N-terminal domain and a C-terminal domain. A narrow, deep cavity accommodating the substrate and cofactor was observed at the junction between the two domains of TcMevK. Computational simulation combined with site-directed mutagenesis and biochemical analyses allowed us to define the binding mode of TcMevK to cofactors and substrates. Moreover, TcMevK showed optimal enzyme activity at pH 8.0 and an optimal temperature of 40 °C for mevalonate as the substrate. The expression profiles and RNA interference of TcMevK indicated its critical role in controlling juvenile hormone biosynthesis, as well as its participation in the production of other terpenoids in T. castaneum. These findings improve our understanding of the structural and biochemical features of insect Mevk and provide a structural basis for the design of MevK inhibitors.

Keywords: enzymatic activity; juvenile hormone biosynthesis; mevalonate pathway.

Figure 1

Structural analysis of TcMevK. (A) Cartoon representation of the TcMevK model in two orientations rotated by 90°. Light blue, N-terminal domain; yellow, C-terminal domain. Secondary elements are labeled. Comparison of the overall structure between TcMevK (gray) and (B) Homo sapiens MevK (PDB ID: 2R3V, slate), (C) Rattus norvegicus MevK (PDB ID: 1KVK, orange), (D) Leishmania major MevK (PDB ID: 2HFS, green), and (E) Methanosarcina mazei MevK (PDB ID: 6MDE, warm pink). All figures were prepared using PyMOL2.5.5. The conformational diversity between TcMevK and its counterparts is highlighted with a dashed frame.

Figure 2

Multiple sequence alignment of TcMevK and other MevK proteins show the conserved cofactor- and substrate-binding site. T. castaneum, Tribolium castaneum MevK (XP_972334.2); D. melanogaster, Drosophila melanogaster MevK (NP_001027412.1); A. thaliana, Arabidopsis thaliana MevK (NP_001190411.1); R. norvegicus, Rattus norvegicus MevK (XP_032742734.1); H. sapiens, Homo sapiens MevK (XP_047284829.1); M. mazei, Methanosarcina mazei MevK (WP_011033702.1); L. major, Leishmania major MevK (XP_001685041.1). The ATP-binding site is labeled as blue stars, and the substrate-binding residues are labeled as red triangles. The conserved GXGXGXX motif was highlighted with a purple frame. Alignments were performed with ClustalW [21] and ESPript [22].

Figure 3

Substrate- and cofactor-binding sites of TcMevK. (A) Binding pattern of cofactor ATP and Magnesium ion to the TcMevK. (B) Binding pattern of substrate Mev to the TcMevK. The residues involved in the cofactor and substrate binding are shown in stick format, docked Mev and ATP are green, and the magnesium ion is magenta. The hydrogen bonds and the salt bridges are yellow dashed lines. The backbone of protein is presented as a semitransparent cartoon.

Figure 4

The enzymatic properties of TcMevK toward Mev. (A) The kinetic parameters of TcMevK toward Mev. (B) The effect of pH on the enzymatic activity of TcMevK. (C) The curve diagrams of enzymatic activity of TcMevK at different temperatures from 20 to 70 °C. Each point represents mean values from three independent tests.

Figure 5

The secondary structure and thermodynamic analyses of TcMevK. (A) The CD spectra of TcMevK at different temperatures from 20 to 90 °C. Conformational changes of α-helix structure at 208 nm (B) and 222 nm (C) in TcMevK induced by temperature. The mean residue ellipticities at 208 nm and 222 nm are used to monitor the conformational changes of TcMevK induced by temperature. (D) The CD spectra of TcMevK at different pH values from 3.0 to 9.0.

Figure 6

Expression patterns and RNA interference of TcMevK in T. castaneum. (A) The expression patterns of TcMevK during the T. castaneum eight key developmental stages at the transcript level. EE, early egg; LE, late egg; EL, early larvae; LL, late larvae; EP, early pupae; LP, late pupae; EA, early adult; LA, late adult. (B) The knockdown efficiency of TcMevK was quantified three and five days after dsRNA injection. (C) The level of Kr-h1 transcript was determined three and five days after TcMevK knockdown. The vertical bars indicate standard errors of the mean (n = 3). Statistically significant differences from the control group are indicated with asterisks: **** p < 0.0001; ** p < 0.01; ns, no significance.