Newly Discovered Penicillin Acylase Activity of Aculeacin A Acylase from
            <i>Actinoplanes utahensis</i>

Torres‐Bacete, Jesús; Hormigo, Daniel; Stuart, M J; Arroyo, Miguel; Torres, Pedro; Castillón, M.P.; Acebal, Carmen; Garcı́a, José Luis; Mata, Isabel de la

doi:10.1128/aem.00452-07

Cited by 23 publications

(24 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…S2 in the supplemental material). Interestingly, these five enzymes show similar substrate specificity with respect to the acyl moiety, because they catalyze the deacylation of substrates with long hydrophobic acyl moieties (4,13,48,49,60,61). In this sense, we have shown in this work that SlPVA is able to hydrolyze not only penicillins but also aculeacin A; in addition, rSlPVA hydrolyzes AHLs (data not shown), raising new questions about the genuine role of SlPVA in nature.…”

Section: Resultsmentioning

confidence: 67%

Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues

Torres‐Bacete

Hormigo

Torres‐Guzmán

et al. 2015

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

bThe pva gene from Streptomyces lavendulae ATCC 13664, encoding a novel penicillin V acylase (SlPVA), has been isolated and characterized. The gene encodes an inactive precursor protein containing a secretion signal peptide that is activated by two internal autoproteolytic cleavages that release a 25-amino-acid linker peptide and two large domains of 18.79 kDa (␣-subunit) and 60.09 kDa (␤-subunit). Based on sequence alignments and the three-dimensional model of SlPVA, the enzyme contains a hydrophobic pocket involved in catalytic activity, including Ser␤1, His␤23, Val␤70, and Asn␤272, which were confirmed by site-directed mutagenesis studies. The heterologous expression of pva in S. lividans led to the production of an extracellularly homogeneous heterodimeric enzyme at a 5-fold higher concentration (959 IU/liter) than in the original host and in a considerably shorter time. According to the catalytic properties of SlPVA, the enzyme must be classified as a new member of the Ntn-hydrolase superfamily, which belongs to a novel subfamily of acylases that recognize substrates with long hydrophobic acyl chains and have biotechnological applications in semisynthetic antifungal production. P enicillin acylase (PA; penicillin amidohydrolase; EC 3.5.1.11) catalyzes the hydrolysis of penicillins into 6-aminopenicillanic acid (6-APA) and the corresponding organic acid. The classification of PAs is based on their substrate specificity, i.e., penicillin G acylases (PGA) or penicillin V acylases (PVA), that preferentially cleave phenylacetyl penicillin (penicillin G [PG]) or phenoxymethyl penicillin (penicillin V [PV]), respectively (1, 2). The relevance of these enzymes lies in the fact that semisynthetic penicillins currently are industrially produced by the enzymatic hydrolysis of PG or PV.PVA is widely distributed among several microorganisms, being intra-and extracellularly produced (2-6). PVA from Streptomyces lavendulae ATCC 13664 (SlPVA) is an extracellular enzyme which has been exhaustively characterized (7-10) and immobilized (11, 12) due to its ability to hydrolyze very efficiently PV and other natural aliphatic penicillins that contaminate PV and usually reduce 6-APA yield at the end of the process. The broad substrate specificity of SlPVA allows this enzyme to hydrolyze several penicillins with aliphatic acyl chains, e.g., 3-hexenoyl-penicillin (penicillin F [PF]), hexanoyl-penicillin (penicillin dihydro-F [PdF]), and octanoyl-penicillin (penicillin K [PK]), as the catalytic constant for PK was even higher than that for PV (13). These observations indicate SlPVA is an effective industrial enzyme, provided that it can be obtained in large amounts.Here, we describe the heterologous overproduction of SlPVA in Streptomyces lividans and the characterization of its catalytic residues by site-directed mutagenesis. MATERIALS AND METHODS Materials.Penicillin V (potassium salt), penicillin G (potassium salt), phenoxyacetic acid, phenylacetic acid, aculeacin A, and fluorescamine were from Sigma-Aldrich (St. Louis, MO). 6-AP...

show abstract

Section: Resultsmentioning

confidence: 67%

Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues

Torres‐Bacete

Hormigo

Torres‐Guzmán

et al. 2015

Appl Environ Microbiol

Self Cite

View full text Add to dashboard Cite

show abstract

“…Most notable are aculeacin acylase from Actinoplanes utahensis (37% identity to PvdQ) (35) and penicillin V acylase from Streptomyces mobaraensis (36% identity) (36). Interestingly, these enzymes have a substrate preference similar to PvdQ.…”

Section: The Catalytic Mechanism Of Pvdq Proceeds Via a Covalently Boundmentioning

confidence: 99%

“…Interestingly, these enzymes have a substrate preference similar to PvdQ. Aculeacin acylase cleaves off long acyl chains, such as linoleic, myristic, or palmitic acid, from the cyclic hexapeptide core of the antifungal echinocandin (35). Penicillin V acylase from Streptomyces mobaraensis has a preference for capsaicin, which consists of an 8-methyl-6-nonene side chain connected to a vanillyl core via a peptide bond.…”

Section: The Catalytic Mechanism Of Pvdq Proceeds Via a Covalently Boundmentioning

confidence: 99%

The quorum-quenching N -acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket

Bokhove¹,

Nadal‐Jimenez

Quax

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

126

163

View full text Add to dashboard Cite

In many Gram-negative pathogens, their virulent behavior is regulated by quorum sensing, in which diffusible signals such as N-acyl homoserine lactones (AHLs) act as chemical messaging compounds. Enzymatic degradation of these diffusible signals by, e.g., lactonases or amidohydrolases abolishes AHL regulated virulence, a process known as quorum quenching. Here we report the first crystal structure of an AHL amidohydrolase, the AHL acylase PvdQ from Pseudomonas aeruginosa. PvdQ has a typical α/β heterodimeric Ntn-hydrolase fold, similar to penicillin G acylase and cephalosporin acylase. However, it has a distinct, unusually large, hydrophobic binding pocket, ideally suited to recognize C12 fatty acid-like chains of AHLs. Binding of a C12 fatty acid or a 3-oxo-C12 fatty acid induces subtle conformational changes to accommodate the aliphatic chain. Furthermore, the structure of a covalent ester intermediate identifies Serβ1 as the nucleophile and Asnβ269 and Valβ70 as the oxyanion hole residues in the AHL degradation process. Our structures show the versatility of the Ntn-hydrolase scaffold and can serve as a structural paradigm for Ntn-hydrolases with similar substrate preference. Finally, the quorum-quenching capabilities of PvdQ may be utilized to suppress the quorum-sensing machinery of pathogens.crystal structure | Pseudomonas aeruginosa | quorum sensing | pyoverdine | catalytic mechanism

show abstract

“…(39) are able to hydrolyze penicillin V but, again, not aliphatic penicillins. However, the acylases from A. utahensis and Streptomyces lavendulae hydrolyze all types of penicillins and show a marked preference for PenK, with K m values 100-to 400-fold smaller than those for PenG (8,12). According to the kinetic data available, the PAC from T. thermophilus would fit the latter category (the category with the acylases from A. utahensis and S. lavendulae) (11).…”

Section: Discussionmentioning

confidence: 99%

“…However, the most thermostable acylases exhibit a preference for aliphatic (penicillins K and dihydroF), rather than aromatic (penicillins G and V), penicillins. For instance, an acylase from the actinomycete Actinoplanes utahensis named aculeacin A acylase, with a T opt of 75°C and t 1/2 at 65°C of 477 min, preferentially hydrolyzes aliphatic penicillins (8,9). A common strategy for finding novel thermostable enzymes (thermozymes) is to screen their microbial diversity, especially among extremophiles, because they have been naturally selected to resist hostile environments.…”

mentioning

confidence: 99%

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

Torres

Cantero

Valle

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position ␣188, ␣189, or ␤24 improved the K m for penicillin G between 9-and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.

show abstract

Newly Discovered Penicillin Acylase Activity of Aculeacin A Acylase from Actinoplanes utahensis

Cited by 23 publications

References 20 publications

Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues

Overexpression of Penicillin V Acylase from Streptomyces lavendulae and Elucidation of Its Catalytic Residues

The quorum-quenching N -acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

Contact Info

Product

Resources

About