Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase
Introduction
Methionine salvage pathway (MSP) is a universal pathway involved in the reprocessing of sulfur-containing cellular metabolites to the amino acid methionine (Sekowska et al., 2004, Albers, 2009). This pathway holds utmost importance owing to the fact that the amount of methionine in the cell is limiting and its de novo synthesis is energetically expensive (Winans and Bassler, 2002, Sekowska et al., 2004). In the MSP, 5-methylthioadenosine (MTA) undergoes a series of steps catalyzed by ∼11 enzymes to form methionine (Sekowska and Danchin, 2002, Ashida et al., 2003). One of these steps involves the isomerization of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via the enzyme 5-methylthioribose 1-phosphate isomerase (M1Pi) (Ashida et al., 2003).
The M1Pi enzymes form a distinct family (No.: PF01008, known as IF-2B family) in the Pfam database (Finn et al., 2015). This family includes two other groups of proteins viz. ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory (α, β and δ) subunits of eukaryotic translation initiation factor 2B (eIF2B) as well. While, the enzyme R15Pi is involved in the nucleoside 5′-monophosphate (NMP) degradation pathway exclusively present in archaea, the eIF2B acts as a guanine nucleotide exchange factor (GEF) during the process of protein translation initiation in eukaryotes; not identified in prokaryotes till date (Price and Proud, 1994, Aono et al., 2012, Nakamura et al., 2012). Although functionally non-related, these three groups of proteins share significant similarity in their primary and tertiary structures (Dev et al., 2009, Gogoi et al., 2016, Kashiwagi et al., 2016). Even though the protomers of these proteins (i.e. members of the PF01008 family) share a common overall topology comprising of N- (α-helical) and C-terminal (αβα-sandwich) domains, their quaternary structures differ. The enzyme R15Pi and the regulatory (α, β and δ) subunits of eIF2B form hexamer (trimer of dimers), whereas the enzyme M1Pi functions as dimer in solution (Bumann et al., 2004, Nakamura et al., 2012, Kashiwagi et al., 2016).
Both the isomerases, M1Pi and R15Pi are known to undergo transition from an open to closed state upon substrate binding mediated by the movement of the N-terminal domain (NTD) towards the C-terminal domain (CTD) (Tamura et al., 2008, Nakamura et al., 2012). The NTD movement upon substrate binding provides a favorable active-site environment for the enzyme reaction to occur (Nakamura et al., 2012). The transition from an open to closed state in these proteins are marked by the formation of a kink in the longest helix of the NTD (Nakamura et al., 2012, Kuhle et al., 2015). Although the domain movement has been proposed for the regulatory subunits (especially for the α-subunit) of eIF2B also (Kuhle et al., 2015, Gogoi and Kanaujia, 2018), no clear evidence has been reported till date.
The enzyme R15Pi catalyzes the conversion of ribose-1,5-bisphosphate (R15P) to ribulose-1,5-bisphosphate (RuBP) via the formation of a cis-phosphoenolate intermediate (Nakamura et al., 2012). However, the catalytic mechanism of the enzyme M1Pi to produce MTRu-1-P from MTR-1-P is not clear. In literature, two different mechanisms of its catalytic action have been suggested (Tamura et al., 2008). One mechanism proceeds via the formation of a cis-phosphoenolate intermediate and is followed by several other isomerases such as triose-phosphate isomerase (TPI), ribose-5-phosphate isomerase (RPI) and R15Pi (Rose and O'Connell, 1960, Rose, 1975, Nakamura et al., 2012). The other mechanism involves a direct hydride transfer assisted by two divalent metal cations as observed in the case of xylose isomerase (XI) (Blow et al., 1992, Asboth and Naray-Szabo, 2000, Fenn et al., 2004). The direct hydride transfer has been suggested as the preferred mechanism of enzyme catalysis for M1Pi due to the lack of incorporation of proton (deuterium) in the product from the medium, a phenomenon common in isomerase enzymes following the cis-enediol mechanism (O'Donoghue et al., 2005a, O'Donoghue et al., 2005b, Berrisford et al., 2006, Saito et al., 2007, Tamura et al., 2008). However, in the case of the enzyme M1Pi, the reaction completes in a metal-independent manner (Saito et al., 2007). Thus, the exact catalytic mechanism of the enzyme M1Pi remains unsettled.
Herein, the three-dimensional crystal structure of M1Pi from Pyrococcus horikoshii OT3 encoded by the open reading frame (ORF) PH0702 has been elucidated. A thorough structural comparison of M1Pi with R15Pi and the regulatory α-subunit of eIF2B to identify and to understand the structural (re)arrangements or the occurrence of structural evolution responsible for their functional specificities have been drawn. In-depth assessment of the microenvironment of the active-site pocket has also been performed to identify the probable preferred mechanism of enzyme reaction followed by M1Pi enzyme.
Section snippets
Cloning, over expression and protein purification
The ORF PH0702 encoding PhM1Pi was PCR-amplified using the forward primer 5′-GCGCGCTAGCATGGAGATAAGATACACTCCAAAGG-3′ and reverse primer 5′-CGACTCGAGTCAAACTGCTTCGTCTCC-3′ containing NheI and XhoI restriction sites (bold), respectively. The amplified gene was inserted into the plasmid pET28a(+) excised with the same set of restriction enzymes. The recombinant plasmid construct was introduced into E. coli Rosetta (DE3) competent cells. Cells were grown at 310 K to an A600 of 0.6 in Luria-Bertani
Overall structure of M1Pi
The crystal structure was solved by molecular replacement method using the atomic coordinates of Archaeoglobus fulgidus (AfM1Pi, PDB id: 1T5O), which shares a sequence identity of 46% with M1Pi from P. horikoshii (hereafter referred to as PhM1Pi). The crystal structure of PhM1Pi was solved in two different forms viz. Form I (P3221) and Form II (P1) which diffracted to a resolution of 2.3 and 2.65 Å, respectively. The asymmetric units (ASU) of the two forms i.e. P3221 and P1 contain two and four
Discussion
The enzyme M1Pi involved in the universally conserved methionine salvage pathway shares high structural similarity with R15Pi and the regulatory (α, β and δ) subunits of eIF2B. Despite this, the proteins M1Pi, R15Pi and eIF2Bα function in completely unrelated cellular processes and show high specificity towards their functions. To perceive the structural features that render functional specificity, a comparison has been drawn among M1Pi, R15Pi and eIF2Bα.
During the methionine salvage pathway,
Accession numbers
Coordinates and structure factors have been deposited in the Protein Data Bank with accession numbers 6A34 and 6A35 for crystal of M1Pi in Form I and Form II, respectively.
Author contributions
SPK conceived the idea of the project; PG and PM performed the experiments; SPK and PG analyzed the results and wrote the paper.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
We express our gratitude to the Central Instruments Facility (CIF), IIT Guwahati for providing access to X-Ray diffractometer. This work was supported by Department of Biotechnology (DBT), Ministry of Science & Technology, Government of India (Project No.: BT/302/NE/TBP/2012).
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