Structure Determination and Characterization of the Vitamin B₆ Degradative Enzyme (E)-2-(Acetamidomethylene)succinate Hydrolase^,

Abstract

The gene identification and kinetic characterization of E-2-(acetamidomethylene)succinate (E-2AMS) hydrolase has recently been described. This enzyme catalyzes the final reaction in the degradation of vitamin B6 and produces succinic semialdehyde, acetate, ammonia, and carbon dioxide from E-2AMS. The structure of E-2AMS hydrolase was determined to 2.3 Å using SAD phasing. E-2AMS hydrolase is a member of the α/β hydrolase superfamily and utilizes a serine/histidine/aspartic acid catalytic triad. Mutation of either the nucleophilic serine or the aspartate resulted in inactive enzyme. Mutation of an additional serine residue in the active site causes the enzyme to be unstable and is likely structurally important. The structure also provides insight into the mechanism of hydrolysis of E-2AMS and identifies several potential catalytically important residues.

Distinct Substrate Selectivity of a Metabolic Hydrolase from Mycobacterium tuberculosis

“…18 Whether this structural plasticity carries over into the structurally similar E-2AMS hydrolases is an interesting area for future investigation, especially as E-2AMS hydrolases already catalyze an unusual chemical conversion. 15 …”

The unusual extended C-terminal helix of the peroxisomal α/β-hydrolase Lpx1 is involved in dimer contacts but dispensable for dimerization

“…The search was carried out with the Dali server (Holm and Rosenstrom, 2010) and resulted in 154 hits with Z-scores above 10, and an rmsd of 2.5-4.1 Å for 174-267 aligned residues despite of a low sequence identity of no more than 20% (Table 2). Similarity is highest between Lpx1 and (E)-2-(acetamidomethylene)succinate (E-2AMS) hydrolase (McCulloch et al, 2010) (Supplementary Fig. S2A).…”

“…We therefore also considered distinct properties of the Lpx1 active site. The Lpx1 active site shows high similarity to the active sites of E-2AMS hydrolase (McCulloch et al, 2010), of an uncharacterized a/b hydrolase (YP_496220.1) from Novosphingobium aromaticivorans (PDB ID 3bwx) and of E. coli YbfF (Park et al, 2008) (Supplementary Fig. S3).…”

“…A Dali search of the PDB 90 for structures similar to the Lpx1 cap (residues 193-267 only) returned three a/b hydrolases among the top five hits. Again, E-2AMS hydrolase (McCulloch et al, 2010) represents the best match, followed by the protein VC1974 from Vibrio cholerae (PDB ID 1r3d), and an a/b hydrolase from Novosphingobium aromaticivorans (PDB ID 3bwx). A search of the PDB with the SSM server (Krissinel and Henrick, 2004) (see Section 2) returned calmodulin as top hit followed by several a/b hydrolases, namely the PPA2-specific methylesterase PME-1 (Xing et al, 2008), the E. coli protein YbfF (Park et al, 2008), bromoperoxidase A2 (Hecht Table 2 Proteins with structural similarities to Lpx1.…”

“…Due to the presence of the additional serine, this has been termed a ''catalytic tetrad'' (Park et al, 2008). However, recent mutagenesis studies suggested that the Ser after strand b7 is required for protein stability (McCulloch et al, 2010). While the acid is a glutamate in Lpx1, it is an aspartate in the other three proteins.…”