Glutamine 89 is a key residue in the allosteric modulation of human serine racemase activity by ATP

Andrea V Canosa,Stefano Armao,Riccardo Percudani,Stefano Bettati,Serena Faggiano,Stefano Bruno,Barbara Campanini,Andrea Mozzarelli,Marialaura Marchetti

doi:10.1038/s41598-018-27227-1

Andrea V Canosa, Stefano Armao + Show 7 more

Open Access

https://doi.org/10.1038/s41598-018-27227-1

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Journal: Scientific Reports	Publication Date: Jun 13, 2018
Citations: 14	License type: open-access

Affiliation: University of Parma, Instituto Biofisika

Abstract

Serine racemase (SR) catalyses two reactions: the reversible racemisation of L-serine and the irreversible dehydration of L- and D-serine to pyruvate and ammonia. SRs are evolutionarily related to serine dehydratases (SDH) and degradative threonine deaminases (TdcB). Most SRs and TdcBs – but not SDHs – are regulated by nucleotides. SR binds ATP cooperatively and the nucleotide allosterically stimulates the serine dehydratase activity of the enzyme. A H-bond network comprising five residues (T52, N86, Q89, E283 and N316) and water molecules connects the active site with the ATP-binding site. Conservation analysis points to Q89 as a key residue for the allosteric communication, since its mutation to either Met or Ala is linked to the loss of control of activity by nucleotides. We verified this hypothesis by introducing the Q89M and Q89A point mutations in the human SR sequence. The allosteric communication between the active site and the allosteric site in both mutants is almost completely abolished. Indeed, the stimulation of the dehydratase activity by ATP is severely diminished and the binding of the nucleotide is no more cooperative. Ancestral state reconstruction suggests that the allosteric control by nucleotides established early in SR evolution and has been maintained in most eukaryotic lineages.

Highlights

Serine racemase (SR) (EC 5.1.1.18) is the enzyme responsible for the synthesis of D-serine, the natural co-agonist of N-methyl-D-aspartate (NMDA) receptors[1]
The residues that form the hydrogen bonding network linking the O3′ of pyridoxal 5′-phosphate (PLP) to the ribose hydroxyl groups of the nucleotide, namely M53, N84, Q87, E281 and N311 in the SpSR structure[14], are identical in the two proteins, with the exception of M53 that is substituted by a Thr residue in StTdcB (Fig. 2A)
PLP-dependent enzymes are renowned for their ability to catalyse secondary reactions in addition to the main one, as is the case for the β-elimination catalysed by transaminases

Summary

Introduction

Serine racemase (SR) (EC 5.1.1.18) is the enzyme responsible for the synthesis of D-serine, the natural co-agonist of N-methyl-D-aspartate (NMDA) receptors[1]. The occupation of the active site by substrates and/or inhibitors leads to conformational changes in the small domain, which rotates with respect to the large domain to restrain accessibility to the active site[14,24] This domain movement, i.e. the transition from an open to a closed structure, is crucial for the racemisation activity. The network connects the O3′ of PLP with the ribose hydroxyl groups and γ-phosphate of AMP-PCP and comprises M53, N84, Q87, E281 and N311 (which correspond to T52, N86, Q89, E283 and N316 in the sequence of hSR) We observed that these residues, with the exception of M53, are present in the close structural homolog of SR bacterial degradative threonine deaminase (TdcB), whose activity is regulated by nucleotides. Phylogenetic analyses and functional characterization allowed to identify Q89 as a key residue in the allosteric communication between the active and allosteric sites of hSR

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