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

Abstract

PDB references: BeSAHase, complex with adenosine and cordycepin, 5m5k; complex with adenine, 5m65; complex with adenosine, 5m66; complex with 2 0 -deoxyadenosine and adenine, 5m67 S-Adenosyl-l-homocysteine hydrolase (SAHase) from the symbiotic bacterium Bradyrhizobium elkanii (BeSAHase) was crystallized in four ligand complexes with (i) mixed adenosine (Ado) and cordycepin (Cord; 3 0 -deoxyadenosine), (ii) adenine (Ade), (iii) Ado and (iv) mixed 2 0 -deoxyadenosine (2 0 -dAdo) and Ade. The crystal structures were solved at resolutions of 1.84, 1.95, 1.95 and 1.54 Å , respectively. Only the Ade complex crystallized with a dimer in the asymmetric unit, while all of the other complexes formed a crystallographically independent tetrameric assembly. In the Ado/Cord complex, adenosine is found in three subunits while the fourth subunit has cordycepin bound in the active site. In the Ade and Ado complexes only these ligand molecules are present in the active sites. The 2 0 -dAdo/Ade complex has Ade bound in two subunits and 2 0 -dAdo bound in the other two subunits. The BeSAHase fold adopted a closed conformation in the complexes with Ado, Ade and 2 0 -dAdo, and a semi-open conformation when cordycepin occupied the active site. An SAHase-specific molecular gate, consisting of residues His342 and Phe343, behaves differently in the different complexes, but there is no simple correlation with the ligand type. Additional small-angle X-ray scattering (SAXS) experiments confirm the tetrameric state of the protein in solution. The main conclusions from this work are (i) that the SAHase subunit does not simply oscillate between two discrete conformational open/closed states in correlation with the absence/presence of a ligand in the active site, but can also assume an intermediate form for some ligands; (ii) that the shut/open state of the molecular gate in the access channel to the active site is not correlated in a simple way with the open/closed subunit conformation or empty/occupied status of the active site, but that a variety of states are possible even for the same ligand; (iii) that a cation (typically sodium) coordinated in an intersubunit loop rigidifies a molecular hinge and thus stabilizes the closed conformation; (iv) that BeSAHase in solution is a tetramer, consistent with the model derived from crystallography.

S-Adenosyl-L-Homocysteine Hydrolase Inhibition by a Synthetic Nicotinamide Cofactor Biomimetic

“…For crystallization, BeSAHase expression and purification was performed as described earlier ( Manszewski et al, 2017 ), using a modified procedure P2, which included protein precipitation with ammonium sulfate as above to remove all ligand molecules bound by the protein at the overexpression step. The only difference to the original P2 procedure was that no NAD + was added to the apo-protein solution.…”

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.…”

“…A very similar coordination geometry of the Na + cation was observed in the crystal structures of other sodium-containing SAHases. [3,6,7,16]…”

Trans‐methylation reactions in plants: focus on the activated methyl cycle

“…Each SAHH monomer is composed of a substrate‐binding domain and a cofactor‐binding domain, where a NAD + molecule is non‐covalently bound. The two domains are separated by a hinge region containing a binding site for cation which rigidifies the hinge structure (Brzezinski et al , Kusakabe et al , Manszewski et al ).…”