Identification and characterization of new family members in the tautomerase superfamily: analysis and implications

Abstract

Tautomerase superfamily members are characterized by a β-α-β building block and a catalytic amino terminal proline. 4-Oxalocrotonate tautomerase (4-OT) and malonate semialdehyde decarboxylase (MSAD) are the title enzymes of two of the five known families in the superfamily. Two recent developments in these families indicate that there might be more metabolic diversity in the tautomerase superfamily than previously thought. 4-OT homologues have been identified in three biosynthetic pathways, whereas all previously characterized 4-OTs are found in catabolic pathways. In the MSAD family, homologues have been characterized that lack decarboxylase activity, but have a modest hydratase activity using 2-oxo-3-pentynoate. This observation stands in contrast to the first characterized MSAD, which is a proficient decarboxylase and a less efficient hydratase. The hydratase activity was thought to be a vestigial and promiscuous activity. However, this recent discovery suggests that the hydratase activity might reflect a new activity in the MSAD family for an unknown substrate. These discoveries open up new avenues of research in the tautomerase superfamily.

Keywords: 4-Oxalocrotonate tautomerase; Catalytic amino terminal proline; Catalytic promiscuity; Hydratase activity; Malonate semialdehyde decarboxylase; Tautomerase superfamily; β–α–β fold.

Figure 1

Ribbon diagrams of the signature monomers in the tautomerase superfamily. A) The 4-oxalocrotonate tautomerase monomer showing a single β–α–β unit (PDB code 4OTA). B) The cis-3-chloroacrylic acid dehalogenase monomer showing the two β–α–β units covalently linked (PDB code 2FLZ). In both structures, Pro-1 is shown in space filling form.

Figure 2

Space filling models showing the active sites of A) 4-oxalocrotonate tautomerase (4-OT) and B) the heterohexamer 4-oxalocrotonate tautomerase (hh4-OT) (PDB codes 4OTA and 3MB2, respectively). In 4-OT, there is space between Phe-50 and Pro-1 (shown as spheres). In the hh4-OT, αTrp-51 fills this space and appears to “crowd” βPro-1 (also shown as spheres).

Figure 3

Sequence Alignment of the Pp and FG41 MSADs along with the five MSAD homologues. Identical residues are indicated by the single asterisk beneath the sequences. Residues that are similar with respect to hydrophobicity/hydrophilicity or charge are marked with · for lower similarity, and : to indicate higher similarity. Pro-1, Asp-37, and Arg-75/76 are conserved in all of the sequences and are highlighted in red. Part of the hydrophobic wall is also highlighted in red. Significant differences in the sequences are described in the text. For clarity, MSAD refers to the Pp MSAD and FG41 refers to the FG41 MSAD. The host organisms for the MSAD homologues are discussed in the text. Alignments were obtained using BLAST and CLUSTAL OMEGA [47,48]

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