Molecular and Catalytic Properties of Monoacetylphloroglucinol Acetyltransferase fromPseudomonassp. YGJ3

Abstract

Monoacetylphloroglucinol (MAPG) acetyltransferase, catalyzing the conversion of MAPG to 2,4-diacetylphloroglucinol (DAPG), was purified from Pseudomonas sp. YGJ3 grown without Cl(-). Cl(-) and pyoluteorin repressed expression of the enzyme. SDS-polyacrylamide gel electrophoresis showed that the purified enzyme (M(r)=330 kDa) was composed of three subunits of 17, 38, and 43 kDa, and protein sequencing identified these as PhlB, PhlA, and PhlC respectively. The enzyme catalyzed the reversible disproportionation of 2 moles of MAPG to phloroglucinol (PG) and DAPG. The equilibrium constant K (=[DAPG][PG]/[MAPG](2)) was estimated to be about 1.0 at 25 °C. A KpnI 20-kb DNA fragment was cloned from the genomic DNA of strain YGJ3, and a 12,598-bp long DNA region containing the phl gene cluster phlACBDEFGHI was sequenced. PCR cloning and expression of the phl genes in Escherichia coli confirmed that expression of phlACB genes produced MAPG ATase.

Distribution of 2,4-Diacetylphloroglucinol Biosynthetic Genes among the Pseudomonas spp. Reveals Unexpected Polyphyletism

“…Gene phlG encodes a hydrolase allowing a return of DAPG to MAPG (Bottiglieri and Keel, 2006). The role of phlI (encoding a hypothetical protein) in DAPG production is still unknown (Hayashi et al, 2012). …”

Molecular cloning, expression, and characterization of acyltransferase from Pseudomonas protegens

“…In contrast to other acyltransferases, the ATase involved in the biosynthesis of DAPG is independent of the coenzyme A (CoA) cofactor (Hayashi et al 2012). No product was detected in the absence of the cell preparations, indicating that product formation can be assigned to the acyltransferase activity present in the mentioned Pseudomonas strains.…”

“…Finally, the operon phlACB encodes an acetyl-CoA independent acyltransferase (ATase), which catalyzes the acetylation of PG leading to the target polyketide DAPG (Shanahan et al 1993; Bangera and Thomashow 1999). Expression of the entire phlACB operon is essential to obtain functional ATase, since individual expression of phlA , phlC , and phlB and subsequent incubation of the three individual proteins did not show any activity towards disproportionation of the natural substrate monoacylphloroglucinol (MAPG) (Bangera and Thomashow 1999; Achkar et al 2005; Hayashi et al 2012). This leads to the assumption that the ATase exists as a multienzyme complex, what was furthermore confirmed by the fact that mutations in any of the genes resulted in a loss of catalytic activity (Bangera and Thomashow 1999; Kidarsa et al 2011).…”

“…YGJ3 was identified to catalyze the reversible disproportionation of two molecules of MAPG ( 6 , Fig. 2) into one molecule of PG ( 7 ) and DAPG ( 5 ) in the forward reaction (Hayashi et al 2012; Yang and Cao 2012; Almario et al 2017). Our previous report proved that a multi-component ATase from Pseudomonas protegens catalyzes transfer of acyl moieties, not only from natural but also from non-natural donor substrates, to the aromatic ring of a phenolic acceptor substrate by forming a new C-C bond in a Friedel-Crafts-type acylation reaction (Schmidt et al 2017).…”

Molecular and Catalytic Properties of 2,4-Diacetylphloroglucinol Hydrolase (PhlG) from <i>Pseudomonas</i> sp. YGJ3

“…The M r of the enzyme was determined by gel filtration with a column (1:8 Â 22 cm) of Superdex 200 (GE Healthcare, Buckinghamshire, UK), which was equilibrated with PM buffer (50 mM potassium phosphate pH 7.0, containing 10 mM 2-mercaptoethanol), and was calibrated with standard proteins as described previously. 9) In a previous study of cloning of the DAPG biosynthetic locus phlHGFACBDEI from Pseudomonas sp. YGJ3 (GenBank accession no.…”