Kinetic, Inhibition, and Structural Characterization of a Malonate Semialdehyde Decarboxylase-like Protein from Calothrix sp. PCC 6303: A Gateway to the non-Pro1 Tautomerase Superfamily Members.

Abstract

The amino-terminal proline (Pro1) has long been thought to be a mechanistic imperative for tautomerase superfamily (TSF) enzymes, functioning as a general base or acid in all characterized reactions. However, a global examination of more than 11,000 nonredundant sequences of the TSF uncovered 346 sequences that lack Pro1. The majority (∼85%) are found in the malonate semialdehyde decarboxylase (MSAD) subgroup where most of the 294 sequences form a separate cluster. Four sequences within this cluster retain Pro1. Because these four sequences might provide clues to assist in the identification and characterization of activities of nearby sequences without Pro1, they were examined by kinetic, inhibition, and crystallographic studies. The most promising of the four (from Calothrix sp. PCC 6303 designated 437) exhibited decarboxylase and tautomerase activities and was covalently modified at Pro1 by 3-bromopropiolate. A crystal structure was obtained for the apo enzyme (2.35 Å resolution). The formation of a 3-oxopropanoate adduct with Pro1 provides clues to build a molecular model for the bound ligand. The modeled ligand extends into a region that allows interactions with three residues (Lys37, Arg56, Glu98), suggesting that these residues can play roles in the observed decarboxylation and tautomerization activities. Moreover, these same residues are conserved in 16 nearby, non-Pro1 sequences in a sequence similarity network. Thus far, these residues have not been implicated in the mechanisms of any other TSF members. The collected observations provide starting points for the characterization of the non-Pro1 sequences.