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.

Ternary complexes of isopentenyl phosphate kinase from Thermococcus paralvinellae reveal molecular determinants of non‐natural substrate specificity

“…The specificity constant k cat /K M and its associated error were calculated using Microsoft Excel (Microsoft Corporation, Redmond, WA, USA). sequence identity with TcpIPK) served as the template, 21 while TCP•DMAP•ADP served as the template for the remaining ternary complexes. Following refinement, the R free value for each structure was 26%-29% for datasets with resolution of 2.5-2.8 Å.…”

“…Further, individual monomers adopted the αβα-sandwich fold common to the AAK superfamily (Figure 3B), resulting in distinct N-(residues 1-166) and C-terminal domains (residues 167-266). 12,21,22 The central β-sheet was composed of eight strands (β3, β6, β2, β1, β9, β13, β14, β12) wedged between bundles of α-helices. One bundle contained three elements (αC, αA, and αH), while the other contained four (αD, αE, αF, αG).…”

“…To date, four homologs have been structurally characterized (Mj, Cma, Tha, Methanothermobacter thermautotrophicus [Mth]), as well as two variants of CmaIPK. 12,21,22 These structures reveal IPKs exist as dimers, with each subunit adopting the αβα sandwich motif common to the AAKs. This fold further divides each monomer into distinct N-and C-terminal domains, with the active site situated in the cleft between them.…”

“…12,20 Moreover, single mutants of the IPK from Candidatus methanomethylophilus alvus (CmaIPK) can accommodate non-natural alkyl-P analogs with linear carbon chains up to 8 atoms in length. 21 This promiscuity has also led to speculation that the isoprenol pathways can be extended to the synthesis of non-natural isoprenoids with potentially useful applications. 19 As such, understanding and tuning the molecular determinants of IPK activity and promiscuity is paramount.…”

“…19 Still, only one homolog (CmaIPK) and its variants have crystal structures bound to non-natural alkyl-P substrates. 21 To gain a more comprehensive understanding of the factors that contribute to the versatility of IPKs, it is essential to investigate additional ternary complexes of IPKs involving non-natural alkyl-P and phosphoryl donor substrates/products. Toward this goal, the current study presents four novel crystal structures of the IPK from Thermococcus paralvinellae (TcpIPK), each in complex with a distinct non-natural phosphoryl acceptor (DMAP; (E)-but-2-en-1-yl monophosphate, ButP; (E)-2-methylbut-2-en-1-yl monophosphate, 2MP; 2-cyclopentylideneethyl monophosphate, CypP; Figure 2) and the phosphoryl donor substrate/product ATP/adenosine diphosphate (ADP).…”