Mutation of βGln114 to Ala Alters the Stabilities of Allosteric States in Tryptophan Synthase Catalysis

Abstract

The tryptophan synthase (TS) bienzyme complexes found in bacteria, yeasts, and molds are pyridoxal 5′-phosphate (PLP)-requiring enzymes that synthesize l-Trp. In the TS catalytic cycle, switching between the open and closed states of the α- and β-subunits via allosteric interactions is key to the efficient conversion of 3-indole-d-glycerol-3′-phosphate and l-Ser to l-Trp. In this process, the roles played by β-site residues proximal to the PLP cofactor have not yet been fully established. βGln114 is one such residue. To explore the roles played by βQ114, we conducted a detailed investigation of the βQ114A mutation on the structure and function of tryptophan synthase. Initial steady-state kinetic and static ultraviolet–visible spectroscopic analyses showed the Q to A mutation impairs catalytic activity and alters the stabilities of intermediates in the β-reaction. Therefore, we conducted X-ray structural and solid-state nuclear magnetic resonance spectroscopic studies to compare the wild-type and βQ114A mutant enzymes. These comparisons establish that the protein structural changes are limited to the Gln to Ala replacement, the loss of hydrogen bonds among the side chains of βGln114, βAsn145, and βArg148, and the inclusion of waters in the cavity created by substitution of the smaller Ala side chain. Because the conformations of the open and closed allosteric states are not changed by the mutation, we hypothesize that the altered properties arise from the lost hydrogen bonds that alter the relative stabilities of the open (βT state) and closed (βR state) conformations of the β-subunit and consequently alter the distribution of intermediates along the β-subunit catalytic path.

Allosteric regulation of substrate channeling: Salmonella typhimurium tryptophan synthase

“…This channeling of the common intermediate, indole, is a key feature of the allosteric control mechanism for the synthesis of L-Trp ( Dunn et al, 1990 ; Miles, 2001 ; Raboni, et al, 2009 ; Dunn, 2012 ; Miles, 2013 ; Hilario et al, 2016 ; Teixeira et al, 2020 ). Within α 2 β 2 , αβ dimers form allosteric units that work independently of each other while the allosteric interactions within each αβ unit are essential to the efficient synthesis of L-Trp ( Dunn, 2012 ; Niks et al, 2013 ; Ghosh et al, 2021 ).…”

“…The TS literature is replete with physical biochemical investigations of the α- and β-reactions ( Yanofsky and Crawford, 1972 ; Miles, 1979 ; Miles, 2001 ; Raboni et al, 2009 ; Dunn, 2012 ; Miles, 2013 ). Recent publications include a variety of solution UV/Vis rapid kinetic studies ( Harris et al, 2005 ; Ngo et al, 2007a , b ; Dierkers et al, 2009 ; Ghosh et al, 2021 ; Phillips and Harris, 2021 ) and NMR studies employing ligands with 19 F ( Niks et al, 2013 ) and 17 O probes ( Young et al, 2016 ). Solid state magic angle spinning ssNMR investigations of TS complexes of substrate and substrate analogues and with 13 C and 15 N enriched PLP ( McDowell et al, 1995 ; Lai et al, 2011 ; Caulkins et al, 2014 , 2016 ; Holmes et al, 2022 ), and β-subunits enriched with 15 N-Lys residues ( Caulkins et al, 2014 ) and molecular dynamics studies ( Huang et al, 2016 ; Maria-Solano et al, 2019 ; O’Rourke et al, 2019 ) have become especially important for establishing the predominant protonation states and tautomeric states of PLP intermediates in the β-reaction.…”

“…As will be discussed in detail in following sections, the switching between open and closed subunit conformations plays a central role in the TS allosteric regulatory mechanism and is critically important to the efficient synthesis of L-Trp ( Dunn et al, 1990 ; Kulik et al, 2005 ; Ngo et al, 2007b ; Barends et al, 2008a ; Lai et al, 2011 ; Niks et al, 2013 ; Caulkins et al, 2016 ; Ghosh et al, 2021 ). Many x-ray crystal structures of the open and closed states of TS are available in the Protein Data Bank archive (RCSB PDB) ( Berman et al, 2000 ).…”

“…Many x-ray crystal structures of the open and closed states of TS are available in the Protein Data Bank archive (RCSB PDB) ( Berman et al, 2000 ). With the high affinity IGP analogue F9 ( 8 ) bound to the α-site, the TS internal aldimine and the L-Ser external aldimine give complexes with closed α-subunits and open β-subunits ( Niks et al, 2013 ; Ghosh et al, 2021 ) (viz, Figure 2 ). The F9 complexes with the α-aminoacrylate and carbanion intermediates almost always give structures where both subunits assume closed conformations ( Ghosh et al, 2021 ).…”

“…With the high affinity IGP analogue F9 ( 8 ) bound to the α-site, the TS internal aldimine and the L-Ser external aldimine give complexes with closed α-subunits and open β-subunits ( Niks et al, 2013 ; Ghosh et al, 2021 ) (viz, Figure 2 ). The F9 complexes with the α-aminoacrylate and carbanion intermediates almost always give structures where both subunits assume closed conformations ( Ghosh et al, 2021 ). 19 F NMR studies of the F9 complexes indicate these subunit conformations are also the predominate forms in solution ( Niks et al, 2013 ).…”