An enzyme captured in two conformational states: crystal structure ofS-adenosyl-L-homocysteine hydrolase fromBradyrhizobium elkanii

Abstract

S-Adenosyl-L-homocysteine hydrolase (SAHase) is involved in the enzymatic regulation of S-adenosyl-L-methionine (SAM)-dependent methylation reactions. After methyl-group transfer from SAM, S-adenosyl-L-homocysteine (SAH) is formed as a byproduct, which in turn is hydrolyzed to adenosine (Ado) and homocysteine (Hcy) by SAHase. The crystal structure of BeSAHase, an SAHase from Bradyrhizobium elkanii, which is a nitrogen-fixing bacterial symbiont of legume plants, was determined at 1.7 Å resolution, showing the domain organization (substrate-binding domain, NAD(+) cofactor-binding domain and dimerization domain) of the subunits. The protein crystallized in its biologically relevant tetrameric form, with three subunits in a closed conformation enforced by complex formation with the Ado product of the enzymatic reaction. The fourth subunit is ligand-free and has an open conformation. The BeSAHase structure therefore provides a unique snapshot of the domain movement of the enzyme induced by the binding of its natural ligands.

Crystallographic and SAXS studies ofS-adenosyl-L-homocysteine hydrolase fromBradyrhizobium elkanii

“…A typical SAHase protomer is usually divided into three closely connected domains: an N-terminal substrate-binding domain, a cofactor-binding domain and a small C-terminal dimerization domain (Brzezinski et al, 2012). The substratebinding and cofactor-binding domains are separated by a bipartite hinge (Manszewski et al, 2015), allowing them to oscillate between two conformational states, closed and open, during the catalytic cycle. The former state is assumed when the domains are close together and is thought to be stabilized by a ligand molecule (substrate, product or inhibitor) bound in the active site (Hu et al, 1999;Yin et al, 2000) and an alkalimetal or ammonium cation coordinated in a metal-binding loop near the active site (Brzezinski et al, 2012;Manszewski et al, 2015).…”

Crystal Structure of S-adenosyl-L-homocysteine Hydrolase from Cytophaga hutchinsonii, a Case of Combination of Crystallographic and Non-crystallographic Symmetry

“…The precise location of the interdomain hinge regions is Since this is not possible for ChSAHase, its linker segments (D184-K195 and H356-V360) were demarcated by sequence alignment with another bacterial SAHase, from Bradyrhizobium elkanii. [8,16] A striking difference between the two tNCS-related intimate dimers AB and CD is visible in their mobility. The AB dimer is less flexible with the mean ADP (atomic displacement parameter) for main-chain atoms of 32.1 Å 2 .…”

“…SAHases are highly conserved proteins, therefore it is not surprising that their interactions with both ligands, as well as with the monovalent cation are very similar among SAHases of various origin. [3,4,[6][7][8][9]11,16] The Mode of Adenosine Binding The substrate-binding site of SAHases is formed by highly conserved amino acid residues. Therefore, the binding mode of Ado is similar to those observed in SAHases of various origin complexed with adenosine or its analogs.…”

“…SAHases usually function as 222-symmetric homotetramers, [4][5][6][7][8][9][10][11] or sometimes as homodimers. [3,12] Each subunit of the enzyme contains a tightly but noncovalently bound nicotinamide adenine dinucleotide cofactor in its oxidized form, NAD + .…”

“…The substrate/product delivery to/from the active site is regulated by a pair of highly conserved His-Phe residues, which function as a molecular gate. [3,7,8] Depending on the conformation of the gate, the entrance to the substratepocket access channel can be open or shut. Additionally, in the closed conformation, the enzyme coordinates a monovalent cation in close proximity of one of the hinge regions and of the active site area.…”