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Dimerization of PHGDH via the catalytic unit is essential for its enzymatic function.

ABSTRACT: Human D-3-phosphoglycerate dehydrogenase (PHGDH), a key enzyme in de novo serine biosynthesis, is amplified in various cancers and serves as a potential target for anticancer drug development. To facilitate this process, more information is needed on the basic biochemistry of this enzyme. For example, PHGDH was found to form tetramers in solution and the structure of its catalytic unit (sPHGDH) was solved as a dimer. However, how the oligomeric states affect PHGDH enzyme activity remains elusive. We studied the dependence of PHGDH enzymatic activity on its oligomeric states. We found that sPHGDH forms a mixture of monomers and dimers in solution with a dimer dissociation constant of ∼0.58 μM, with the enzyme activity depending on the dimer content. We computationally identified hotspot residues at the sPHGDH dimer interface. Single-point mutants at these sites disrupt dimer formation and abolish enzyme activity. Molecular dynamics simulations showed that dimer formation facilitates substrate binding and maintains the correct conformation required for enzyme catalysis. We further showed that the full-length PHGDH exists as a dynamic mixture of monomers, dimers, and tetramers in solution with enzyme concentration-dependent activity. Mutations that can completely disrupt the sPHGDH dimer show different abilities to interrupt the full-length PHGDH tetramer. Among them, E108A and I121A can also disrupt the oligomeric structures of the full-length PHGDH and abolish its enzyme activity. Our study indicates that disrupting the oligomeric structure of PHGDH serves as a novel strategy for PHGDH drug design and the hotspot residues identified can guide the design process.

SUBMITTER: Xu H

PROVIDER: S-EPMC8081924 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:PDE4 family cAMP phosphodiesterases play a pivotal role in determining compartmentalised cAMP signalling through targeted cAMP breakdown. Expressing the widely found PDE4D5 isoform, as both bait and prey in a yeast 2-hybrid system, we demonstrated interaction consistent with the notion that long PDE4 isoforms form dimers. Four potential dimerization sites were uncovered using a scanning peptide array approach, where a recombinant purified PDE4D5 fusion protein was used to probe a 25-mer library of overlapping peptides covering the entire PDE4D5 sequence. Key residues involved in PDE4D5 dimerization were defined using a site-directed mutagenesis programme directed by an alanine scanning peptide array approach. Critical residues stabilising PDE4D5 dimerization were defined within the regulatory UCR1 region found in long, but not short, PDE4 isoforms, namely the Arg(173), Asn(174) and Asn(175) (DD1) cluster. Disruption of the DD1 cluster was not sufficient, in itself, to destabilise PDE4D5 homodimers. Instead, disruption of an additional interface, located on the PDE4 catalytic unit, was also required to convert PDE4D5 into a monomeric form. This second dimerization site on the conserved PDE4 catalytic unit is dependent upon a critical ion pair interaction. This involves Asp(463) and Arg(499) in PDE4D5, which interact in a trans fashion involving the two PDE4D5 molecules participating in the homodimer. PDE4 long isoforms adopt a dimeric state in living cells that is underpinned by two key contributory interactions, one involving the UCR modules and one involving an interface on the core catalytic domain. We propose that short forms do not adopt a dimeric configuration because, in the absence of the UCR1 module, residual engagement of the remaining core catalytic domain interface provides insufficient free energy to drive dimerization. The functioning of PDE4 long and short forms is thus poised to be inherently distinct due to this difference in quaternary structure.

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Dimerization of PHGDH via the catalytic unit is essential for its enzymatic function.

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