Biochemical and Structural Analysis of RraA Proteins To Decipher Their Relationships with 4‑Hydroxy-4-methyl-2-oxoglutarate/4-Carboxy-4-hydroxy-2-oxoadipate Aldolases

Abstract

4-Hydroxy-4-methyl-2-oxoglutarate (HMG)/4-carboxy-4-hydroxy-2-oxoadipate (CHA) aldolases are class II (divalent metal ion dependent) pyruvate aldolases from the <i>meta</i> cleavage pathways of protocatechuate and gallate. The enzyme from <i>Pseudomonas putida</i> F1 is structurally similar to a group of proteins termed regulators of RNase E activity A (RraA) that bind to the regulatory domain of RNase E and inhibit the ribonuclease activity in certain bacteria. Analysis of homologous RraA-like proteins from varying species revealed that they share sequence conservation within the active site of HMG/CHA aldolase. In particular, the <i>P. putida</i> F1 HMG/CHA aldolase has a D-X₂₀-R-D motif, whereas a G-X₂₀-R-D-X₂-E/D motif is observed in the structures of the RraA-like proteins from <i>Thermus thermophilus</i> HB8 (<i>Tt</i>RraA) and <i>Saccharomyces cerevisiae</i> S288C (Yer010Cp) that may support metal binding. <i>Tt</i>RraA and Yer010Cp were found to contain HMG aldolase and oxaloacetate decarboxylase activities. Similar to the <i>P. putida</i> F1 HMG/CHA aldolase, both <i>Tt</i>RraA and Yer010Cp enzymes required divalent metal ions for activity and were competitively inhibited by oxalate, a pyruvate enolate analogue, suggesting a common mechanism among the enzymes. The RraA from <i>Escherichia coli</i> (<i>Ec</i>RraA) lacked detectable C–C lyase activity. Upon restoration of the G-X₂₀-R-D-X₂-E/D motif, by site-specific mutagenesis, the <i>Ec</i>RraA variant was able to catalyze oxaloacetate decarboxylation. Sequence analysis of RraA-like gene products found across all the domains of life revealed conservation of the metal binding motifs that can likely support a divalent metal ion-dependent enzyme reaction either in addition to or in place of the putative RraA function