The Crystal Structure of YdcE, a 4-Oxalocrotonate Tautomerase Homologue from Escherichia coli, Confirms the Structural Basis for Oligomer Diversity^,

Abstract

The tautomerase superfamily consists of three major families represented by 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), and macrophage migration inhibitory factor (MIF). The members of this superfamily are structurally homologous proteins constructed from a simple beta-alpha-beta fold that share a key mechanistic feature; they use an amino-terminal proline, which has an unusually low pK(a), as the general base in a keto-enol tautomerization. Several new members of the 4-OT family have now been identified using PSI-BLAST and categorized into five subfamilies on the basis of multiple-sequence alignments and the conservation of key catalytic and structural residues. The members of subfamily 5, which includes a hypothetical protein designated YdcE from Escherichia coli, are predicted not to form hexamers. The crystal structure of YdcE has been determined to 1.35 A resolution and confirms that it is a dimer. In addition, YdcE complexed with (E)-2-fluoro-p-hydroxycinnamate, identified as a potent competitive inhibitor of this enzyme, as well as N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) and benzoate are also presented. These latter crystal structures reveal the location of the active site and suggest a mechanism for the observed YdcE-catalyzed tautomerization reaction. The dimeric arrangement of YdcE represents a new structure in the 4-OT family and demonstrates structural diversity within the 4-OT family not previously reported.

Crystal Structures of Native and Inactivated cis-3-Chloroacrylic Acid Dehalogenase

“…The other four families, represented by their title enzymes, are the 5-(carboxymethyl)-2-hydroxymuconate isomerase (28), 4-oxalocrotonate tautomerase (26,29), macrophage migration inhibitory factor (30), and malonate semialdehyde decarboxylase (MSAD) families (27). cis-CaaD has been classified in a separate tautomerase family, the cis-CaaD family, because of the absence of significant sequence identity with known members of the other four families (1).…”

Mechanistic Characterization of a Bacterial Malonate Semialdehyde Decarboxylase

“…Several 4-OT homologues have been assigned to the 4-OT family of enzymes on the basis of sequence analysis (15). These homologues are composed of small subunits (typically 61-81 amino acid residues) and have a conserved N-terminal proline.…”

Kinetic and Stereochemical Analysis of YwhB, a 4-Oxalocrotonate Tautomerase Homologue in Bacillus subtilis: Mechanistic Implications for the YwhB- and 4-Oxalocrotonate Tautomerase-Catalyzed Reactions

“…The tautomerase superfamily is a group of structurally homologous enzymes sharing a characteristic β-α-β building block and a catalytic Pro-1 (16,(28)(29)(30). The 4-OT family, named for the first characterized enzyme in the family, is one of the five known families in this superfamily.…”

“…The 4-OT family, named for the first characterized enzyme in the family, is one of the five known families in this superfamily. 4-OT family members, including YwhB, are typically constructed from short sequences (61-84 amino acids) and are found throughout the microbial community (29). Only a handful of these family members have assigned functions (29,30).…”

“…4-OT family members, including YwhB, are typically constructed from short sequences (61-84 amino acids) and are found throughout the microbial community (29). Only a handful of these family members have assigned functions (29,30). The inability to assign functions for the majority of the family members precludes an understanding of this niche in microbial metabolism.…”