Molecular Basis for the Substrate Promiscuity of Isopentenyl Phosphate Kinase from Candidatus methanomethylophilus alvus

Abstract

Isopentenyl phosphate kinases (IPKs) catalyze the ATP-dependent phosphorylation of isopentenyl monophosphate (IP) to isopentenyl diphosphate (IPP) in the alternate mevalonate pathways of the archaea and plant cytoplasm. In recent years, IPKs have also been employed in artificial biosynthetic pathways called "(iso) prenol pathways" that utilize promiscuous kinases to sequentially phosphorylate (iso) prenol and generate the isoprenoid precursors IPP and dimethylallyl diphosphate (DMAPP). Furthermore, IPKs have garnered attention for their impressive substrate promiscuity toward non-natural alkyl-monophosphates (alkyl-Ps), which has prompted their utilization as biocatalysts for the generation of novel isoprenoids. However, none of the IPK crystal structures currently available contain non-natural substrates, leaving the roles of active-site residues in substrate promiscuity ambiguous. To address this, we present herein the high-resolution crystal structures of an IPK from Candidatus methanomethylophilus alvus (CMA) in the apo form and bound to natural and non-natural substrates. Additionally, we describe active-site engineering studies leading to enzyme variants with broadened substrate scope, as well as structure determination of two such variants (Ile74Ala and Ile146Ala) bound to non-natural alkyl-Ps. Collectively, our crystallographic studies compare six structures of CMA variants in different ligand-bound forms and highlight contrasting structural dynamics of the two substrate-binding sites. Furthermore, the structural and mutational studies confirm a novel role of the highly conserved DVTGG motif in catalysis, both in CMA and in IPKs at large. As such, the current study provides a molecular basis for the substrate-binding modes and catalytic performance of CMA toward the goal of developing IPKs into useful biocatalysts.

Figure 1.

Mevalonate pathways found in nature. The bifurcation of the general scheme occurs in the final two steps, where the diphosphorylation occurs either before (classical, solid box) or after the decarboxylation (alternate, dashed box). Abbreviations: CoA = coenzyme A, AAT = acetoacetyl-CoA thiolase, HMGS = 3-hydroxy-3-methyl-glutaryl-CoA synthase, HMGR = 3-hydroxy-3-methyl-glutaryl-CoA reductase, M5K = mevalonate 5-kinase, PHMK = phosphomevalonate kinase, PMD = phosphomevalonate decarboxylase, DPMD = diphosphomevalonate decarboxylase, IPK = isopentenyl phosphate kinase, and IDI = isopentenyl diphosphate isomerase.

Figure 2.

Overall structure of CMA•IP•ATP. The dimer interface is visualized in (A) with Chain A shown in green and Chain B in red. Monomer B is highlighted in (B) with the secondary structure elements colored red (α helices), yellow (β strands), and green (3₁₀ helices). This color scheme is mirrored in the topology map depicted in (C). The ligands in (A) and (B) are shown in cyan (IP/ADP) and yellow (IPP/ATP).

Figure 3.

Active sites of CMA•IP•ATP and CMA•BP•ADP. The interactions of IP with ATP/ADP across monomers A and B of CMA•IP•ATP are depicted in (A). The carbon atoms of IP are colored yellow/dark blue, the carbon atoms of ATP/ADP are colored blue/green, phosphates are colored green/orange and sky blue/red, water molecules are modeled as light orange/cyan spheres, active site residues are colored gray/lime, and important H-bonds are shown with black/magenta dashed lines (for monomer A/B, respectively). The interactions of IPP and ADP in monomer A of CMA•IP•ATP are depicted in (B). The carbon atoms of IPP and ATP are colored yellow and green, respectively; active site residues are colored gray; and water molecules are modeled as light orange spheres. Important H-bonds are shown with black dashed lines. The active site of CMA•BP•ADP monomer B overlaid with IP is depicted in (C). The carbon atoms of IP, ADP, and BP are colored yellow, green, and magenta (respectively); active-site residues are colored gray; and water molecules are modeled as light orange spheres. Important H-bonds are shown with black dashed lines.

Figure 4.

(A) Library of alkyl-P analogues utilized for the current study. (B) Results of screening CMA variants against the library of alkyl-P analogues using the PK-LDH assay. Conversions were calculated by measuring the absorbance at 340 nm of each enzymatic reaction just before and 1 h after the addition of the CMA variant at 37 °C and comparing it to a positive control (n = 2). Appropriate controls were conducted to account for any ATPase activity. Each reaction consisted of 2 U PK, 2 U LDH, 0.6 mM NADH, 1.5 mM PEP, 2 mM ATP, and 4 μg of IPK incubated in a buffered solution (25 mM Tris pH 7.8 and 5 mM MgCl₂). All positive reactions were verified using high-resolution mass spectrometry (HRMS). Conversion data for WT CMA were obtained previously.

Figure 5.

Alkyl-binding sites of I74A•38•ADP and I146A•26-P•ADP. The interactions of 38 and 26-P are depicted in (A) and (B), respectively. The carbon atoms of 38 and 26-P are depicted in yellow and magenta (respectively), water molecules are modeled as yellow or orange spheres, and important polar contacts are shown with black dashed lines.

Figure 6.

Comparison of the orientations of 38 (A), 26 (B), 26-P (C), and 27 (D) with their natural comparators IP and IPP in the binding pockets of CMA variants. The carbons of 38, 26/26-P, and 27 are colored cyan, purple, and tan (respectively), while their phosphate groups are shown in orange and red. The carbons of IP and IPP are colored yellow, while their phosphate groups are shown in green and sky blue.

Figure 7.

(A) Nucleotide-binding site of monomer A in I74A•38•ADP. The carbons of ADP are colored green, water molecules are modeled as yellow spheres, and important polar contacts are shown with black dashed lines. (B) Conformational changes of the nucleotide-binding domain of the WT apo structure (red) and the I146A•27•ADP (green) and I74A•38•ADP (yellow) complexes.

Figure 8.

Proposed Mg²⁺-binding site in I74A•38•ADP in monomer A. The water modeled in place of Mg²⁺ is colored dark green, while the remaining waters are shown as yellow stars. The carbons of 38 and ADP are colored green and purple (respectively), active-site residues are colored teal, and important polar contacts are shown with black dashed lines.

Figure 9.

Results of screening DVTGG variants of CMA WT (A) and I74A (B) against the library of alkyl-P analogues using the PK-LDH assay. Conversions were calculated by measuring the absorbance at 340 nm of each enzymatic reaction just before and 1 h after the addition of the CMA variant at 37 °C and comparing it to a positive control (n = 2). Appropriate controls were conducted to account for any ATPase activity. Each reaction consisted of 2 U PK, 2 U LDH, 0.6 mM NADH, 1.5 mM PEP, 2 mM ATP, and 4 μg of IPK incubated in a buffered solution (25 mM Tris pH 7.8 and 5 mM MgCl₂). All positive reactions were verified using HRMS. Conversion data for WT CMA were obtained previously.