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Structural insight into substrate and product binding in an archaeal mevalonate kinase.

ABSTRACT: Mevalonate kinase (MK) is a key enzyme of the mevalonate pathway, which produces the biosynthetic precursors for steroids, including cholesterol, and isoprenoids, the largest class of natural products. Currently available crystal structures of MK from different organisms depict the enzyme in its unbound, substrate-bound, and inhibitor-bound forms; however, until now no structure has yet been determined of MK bound to its product, 5-phosphomevalonate. Here, we present crystal structures of mevalonate-bound and 5-phosphomevalonate-bound MK from Methanosarcina mazei (MmMK), a methanogenic archaeon. In contrast to the prior structure of a eukaryotic MK bound with mevalonate, we find a striking lack of direct interactions between this archaeal MK and its substrate. Further, these two MmMK structures join the prior structure of the apoenzyme to complete the first suite of structural snapshots that depict unbound, substrate-bound, and product-bound forms of the same MK. With this collection of structures, we now provide additional insight into the catalytic mechanism of this biologically essential enzyme.

SUBMITTER: Miller BR

PROVIDER: S-EPMC6283576 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Structural insight into substrate and product binding in an archaeal mevalonate kinase.

Miller Bradley R BR Kung Yan Y

PloS one 20181206 12

Mevalonate kinase (MK) is a key enzyme of the mevalonate pathway, which produces the biosynthetic precursors for steroids, including cholesterol, and isoprenoids, the largest class of natural products. Currently available crystal structures of MK from different organisms depict the enzyme in its unbound, substrate-bound, and inhibitor-bound forms; however, until now no structure has yet been determined of MK bound to its product, 5-phosphomevalonate. Here, we present crystal structures of mevalo ...[more]

PMID: 30521590

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Project description:BackgroundIsoprenoid precursor synthesis via the mevalonate route in humans and pathogenic trypanosomatids is an important metabolic pathway. There is however, only limited information available on the structure and reactivity of the component enzymes in trypanosomatids. Since isoprenoid biosynthesis is essential for trypanosomatid viability and may provide new targets for therapeutic intervention it is important to characterize the pathway components.ResultsPutative mevalonate kinase encoding genes from Leishmania major (LmMK) and Trypanosoma brucei (TbMK) have been cloned, over-expressed in and proteins isolated from procyclic-form T. brucei. A highly sensitive radioactive assay was developed and shows ATP-dependent phosphorylation of mevalonate. Apo and (R)-mevalonate bound crystal structures of LmMK, from a bacterial expression system, have been determined to high resolution providing, for the first time, information concerning binding of mevalonate to an MK. The mevalonate binds in a deep cavity lined by highly conserved residues. His25 is key for binding and for discrimination of (R)- over (S)-mevalonate, with the main chain amide interacting with the C3 hydroxyl group of (R)-mevalonate, and the side chain contributing, together with Val202 and Thr283, to the construction of a hydrophobic binding site for the C3 methyl substituent. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. The activity of LmMK was significantly reduced compared to MK from other species and we were unable to obtain ATP-binding data. Comparisons with the rat MK:ATP complex were used to investigate how this substrate might bind. In LmMK, helix alpha2 and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding.ConclusionOur new structural information, consistent with data on homologous enzymes allows a detailed description of how mevalonate is recognized and positioned for catalysis in MK. The mevalonate-binding site is highly conserved yet the ATP-binding site is structurally distinct in LmMK. We are unable to provide a definitive explanation for the low activity of recombinant protein isolated from a bacterial expression system compared to material isolated from procyclic-form Trypanosoma brucei.

Project description:The alkaline α-galactosidase AtAkαGal3 from Arabidopsis thaliana catalyzes the hydrolysis of α-D-galactose from galacto-oligosaccharides under alkaline conditions. A phylogenetic analysis based on sequence alignment classifies AtAkαGal3 as more closely related to the raffinose family of oligosaccharide (RFO) synthases than to the acidic α-galactosidases. Here, thin-layer chromatography is used to demonstrate that AtAkαGal3 exhibits a dual function and is capable of synthesizing stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkαGal3 and its D383A mutant with various substrates and products, including galactose, galactinol, raffinose, stachyose and sucrose, are reported as the first representative structures of an alkaline α-galactosidase. The structure of AtAkαGal3 comprises three domains: an N-terminal domain with 13 antiparallel β-strands, a catalytic domain with an (α/β)8-barrel fold and a C-terminal domain composed of β-sheets that form two Greek-key motifs. The WW box of the N-terminal domain, which comprises the conserved residues FRSK75XW77W78 in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (α/β)8 barrel of the catalytic domain. The C-terminal domain is presumably involved in structural stability. Structures of the D383A mutant in complex with various substrates and products, especially the natural substrate/product stachyose, reveal four complete subsites (-1 to +3) at the catalytic site. A functional loop (residues 329-352) that exists in the alkaline α-galactosidase AtAkαGal3 and possibly in RFO synthases, but not in acidic α-galactosidases, stabilizes the stachyose at the +2 and +3 subsites and extends the catalytic pocket for the transferase mechanism. Considering the similarities in amino-acid sequence, catalytic domain and activity between alkaline α-galactosidases and RFO synthases, the structure of AtAkαGal3 might also serve a model for the study of RFO synthases, structures of which are lacking.

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Structural insight into substrate and product binding in an archaeal mevalonate kinase.

Publications

Structural insight into substrate and product binding in an archaeal mevalonate kinase.

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