Sbi00515, a Protein of Unknown Function from Streptomyces bingchenggensis, Highlights the Functional Versatility of the Acetoacetate Decarboxylase Scaffold

Abstract

The acetoacetate decarboxylase-like superfamily (ADCSF) is a group of ~4000 enzymes that, until recently, was thought to be homogeneous in terms of the reaction catalyzed. Bioinformatic analysis shows that the ADCSF consists of up to seven families that differ primarily in their active site architectures. The soil-dwelling bacterium Streptomyces bingchenggensis BCW-1 produces an ADCSF enzyme of unknown function that shares a low level of sequence identity (~20%) with known acetoacetate decarboxylases (ADCs). This enzyme, Sbi00515, belongs to the MppR-like family of the ADCSF because of its similarity to the mannopeptimycin biosynthetic protein MppR from Streptomyces hygroscopicus. Herein, we present steady state kinetic data that show Sbi00515 does not catalyze the decarboxylation of any α- or β-keto acid tested. Rather, we show that Sbi00515 catalyzes the condensation of pyruvate with a number of aldehydes, followed by dehydration of the presumed aldol intermediate. Thus, Sbi00515 is a pyruvate aldolase-dehydratase and not an acetoacetate decarboxylase. We have also determined the X-ray crystal structures of Sbi00515 in complexes with formate and pyruvate. The structures show that the overall fold of Sbi00515 is nearly identical to those of both ADC and MppR. The pyruvate complex is trapped as the Schiff base, providing evidence that the Schiff base chemistry that drives the acetoacetate decarboxylases has been co-opted to perform a new function, and that this core chemistry may be conserved across the superfamily. The structures also suggest possible catalytic roles for several active site residues.

Swit_4259, an acetoacetate decarboxylase-like enzyme from Sphingomonas wittichii RW1

“…As shown in Fig. 2(a), the overall fold of the Swit_4259 protomer is nearly identical to that of the proto-typical family I ADCs, as well as the recently described family V enzymes such as SbAD from Streptomyces bingchenggensis and MppR from S. hygroscopicus (not shown), which are not decarboxylases (Burroughs et al, 2013;Mueller et al, 2015). This fold, dubbed the -cone (Ho et al, 2009) or double-barrel fold (Tagaki & Westheimer, 1968), consists of a large, antiparallel, 13-stranded -sheet that forms the main barrel and folds back on itself to form a second, orthogonal barrel (Fig.…”

“…The high degree of structural similarity between the MppR, SbAD and Swit_4259 active sites suggested that the latter enzyme would also accept an -keto acid as a substrate. Our previous work on SbAD found that Tyr252 plays a key role in the aldolasedehydratase activity of this enzyme (Mueller et al, 2015). Likewise, the equivalent residue in MppR, Glu283, appears to be important for its cyclization reaction (Burroughs et al, 2013).…”

“…5a) was qualitatively modelled into the Swit_4259 active site (Fig. 6a) based on an overlay with the pyruvate-bound structure of SbAD (PDB entry 4zbt; Mueller et al, 2015). While not based on experimental data, this model did suggest that -keto dicarboxylic acids of sufficient length are likely to bind to the enzyme.…”

“…Despite relatively low amino-acid sequence identity to the prototypical Clostridium acetobutylicum acetoacetate decarboxylase (AAB53232; 18%), Swit_4259 is identified by the NCBI Conserved Domain Database (CDD) as a member of the acetoacetate decarboxylase-like superfamily (ADCSF; PF06314). The ADCSF is an incompletely characterized group of enzymes with significant diversity in both substrate and reaction specificities (Burroughs et al, 2013;Mueller et al, 2015). Based on sequence alignments and manual interrogation of putative active-site residues, the ADCSF can be divided into seven families (Burroughs et al, 2013).…”

“…S1). Another related enzyme, the aldolase-dehydratase from S. bingchenggensis (SbAD), was shown to be an efficient catalyst of the aldol condensation of pyruvate with a number of unsaturated aliphatic and aromatic aldehydes (Mueller et al, 2015; Supplementary Fig. S1).…”