Mechanistic Studies of the Streptomyces bingchenggensis Aldolase-Dehydratase: Implications for Substrate and Reaction Specificity in the Acetoacetate Decarboxylase-like Superfamily.

The acetoacetate decarboxylase-like superfamily (ADCSF) is a little-explored group of enzymes that may contain new biocatalysts. The low level of sequence identity (∼20%) between many ADCSF enzymes and the confirmed acetoacetate decarboxylases led us to investigate the degree of diversity in the reaction and substrate specificity of ADCSF enzymes. We have previously reported on Sbi00515, which belongs to Family V of the ADCSF and functions as an aldolase-dehydratase. Here, we more thoroughly characterize the substrate specificity of Sbi00515 and find that aromatic, unsaturated aldehydes yield lower KM and higher kcat values compared to those of other small electrophilic substrates in the condensation reaction. The roles of several active site residues were explored by site-directed mutagenesis and steady state kinetics. The lysine-glutamate catalytic dyad, conserved throughout the ADCSF, is required for catalysis. Tyrosine 252, which is unique to Sbi00515, is hypothesized to orient the incoming aldehyde in the condensation reaction. Transient state kinetics and an intermediate-bound crystal structure aid in completing a proposed mechanism for Sbi00515.

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

The Gram-negative bacterium Sphingomonas wittichii RW1 is notable for its ability to metabolize a variety of aromatic hydrocarbons. Not surprisingly, the S. wittichii genome contains a number of putative aromatic hydrocarbon-degrading gene clusters. One of these includes an enzyme of unknown function, Swit_4259, which belongs to the acetoacetate decarboxylase-like superfamily (ADCSF). Here, it is reported that Swit_4259 is a small (28.8 kDa) tetrameric ADCSF enzyme that, unlike the prototypical members of the superfamily, does not have acetoacetate decarboxylase activity. Structural characterization shows that the tertiary structure of Swit_4259 is nearly identical to that of the true decarboxylases, but there are important differences in the fine structure of the Swit_4259 active site that lead to a divergence in function. In addition, it is shown that while it is a poor substrate, Swit_4259 can catalyze the hydration of 2-oxo-hex-3-enedioate to yield 2-oxo-4-hydroxyhexanedioate. It is also demonstrated that Swit_4259 has pyruvate aldolase-dehydratase activity, a feature that is common to all of the family V ADCSF enzymes studied to date. The enzymatic activity, together with the genomic context, suggests that Swit_4259 may be a hydratase with a role in the metabolism of an as-yet-unknown hydrocarbon. These data have implications for engineering bioremediation pathways to degrade specific pollutants, as well as structure-function relationships within the ADCSF in general.

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

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 ß-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.

Structural and functional characterization of MppR, an enduracididine biosynthetic enzyme from streptomyces hygroscopicus: functional diversity in the acetoacetate decarboxylase-like superfamily.

The nonproteinogenic amino acid enduracididine is a critical component of the mannopeptimycins, cyclic glycopeptide antibiotics with activity against drug-resistant pathogens, including methicillin-resistant Staphylococcus aureus. Enduracididine is produced in Streptomyces hygroscopicus by three enzymes, MppP, MppQ, and MppR. On the basis of primary sequence analysis, MppP and MppQ are pyridoxal 5'-phosphate-dependent aminotransferases; MppR shares a low, but significant, level of sequence identity with acetoacetate decarboxylase. The exact reactions catalyzed by each enzyme and the intermediates involved in the route to enduracididine are currently unknown. Herein, we present biochemical and structural characterization of MppR that demonstrates a catalytic activity for this enzyme and provides clues about its role in enduracididine biosynthesis. Bioinformatic analysis shows that MppR belongs to a previously uncharacterized family within the acetoacetate decarboxylase-like superfamily (ADCSF) and suggests that MppR-like enzymes may catalyze reactions diverging from the well-characterized, prototypical ADCSF decarboxylase activity. MppR shares a high degree of structural similarity with acetoacetate decarboxylase, though the respective quaternary structures differ markedly and structural differences in the active site explain the observed loss of decarboxylase activity. The crystal structure of MppR in the presence of a mixture of pyruvate and 4-imidazolecarboxaldehyde shows that MppR catalyzes the aldol condensation of these compounds and subsequent dehydration. Surprisingly, the structure of MppR in the presence of "4-hydroxy-2-ketoarginine" shows the correct 4R enantiomer of "2-ketoenduracididine" bound to the enzyme. These data, together with bioinformatic analysis of MppR homologues, identify a novel family within the acetoacetate decarboxylase-like superfamily with divergent active site structure and, consequently, biochemical function.

A concise synthesis of (+/-)-cacalol.

A simple synthesis of the natural product cacalol has been developed that proceeds in seven steps and 21-25% overall yield. Ortho-lithiation of 4-methylanisole and alkylation with 5-iodo-1-pentene, followed by intramolecular Friedel-Crafts alkylation, gave 5-methoxy-1,8-dimethyltetralin. This compound was then formylated in the 6-position. Baeyer-Villiger oxidation and hydrolysis of the resulting formate gave 6-hydroxy-5-methoxy-1,8-dimethyltetralin. Alkylation of the phenolic hydroxyl group with chloroacetone followed by cyclodehydration gave cacalol methyl ether. Deprotection of this aryl methyl ether yielded cacalol.