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

Aculeacin A acylase from Actinoplanes utahensis produced byStreptomyces lividans revealed acylase activities that are ableto hydrolyze penicillin V and several natural aliphatic penicillins.Penicillin K was the best substrate, showing a catalytic efficiencyof 34.79 mM^–1 s^–1. Furthermore, aculeacin A acylasewas highly thermostable, with a midpoint transition temperatureof 81.5°C.

INTRODUCTION

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INTRODUCTION

Aculeacin A acylase (EC 3.5.1.70) from Actinoplanes utahensisNRRL 12052 (AuAAC) catalyzes the hydrolyses of the acyl moietiesof antifungal echinocandin antibiotics produced by Aspergillusspecies (13) (Fig. 1A). These cyclic hexapeptide antibioticscontain long fatty acid side chains (e.g., linoleic, myristic,or palmitic acid) and exhibit high antifungal activity. Theyhave been used to produce several potential therapeutic agentsafter enzymatic hydrolysis of their acidic moieties, which releasesa cyclic hexapeptide that can be further reacylated (3, 6).

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FIG. 1. (A) Hydrolysis of aculeacin A catalyzed by AAC from A. utahensis. (B) Hydrolysis of penicillin V by penicillin V acylase. The small arrows indicate the positions of hydrolysis.

AuAAC is largely extracellular and consists of two dissimilarsubunits with molecular masses of 60 kDa and 19 kDa (8, 18).Remarkably, although this acylase shares sequence similaritywith ß-lactam acylases (8, 11), it has not been reportedto show penicillin acylase activity. Preliminary analyses carriedout in our laboratory showed that AuAAC was very similar tothe ß-lactam acylase from Streptomyces lavendulae(AY611030), which shows a preference for penicillins with longhydrophobic acyl moieties (21). On the other hand, althoughAuAAC has been purified (8, 9, 18), few data on its structurefunction relationships have been provided so far and many biochemicalproperties of these proteins remain to be analyzed.

Taking into account these observations, we have investigatedwhether AuAAC would behave as a new ß-lactam acylasewith novel properties. In this work, we describe newly discoveredhydrolytic activities of AuAAC overproduced and purified froma recombinant Streptomyces lividans strain harboring the aacgene.

Overproduction of AuAAC.

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Overproduction of AuAAC.

To overproduce AuAAC, an engineered aac gene was constructedand cloned into the bifunctional expression vector pEM4, whichcontains the ermE* promoter from Saccharopolyspora erythraea(15). The aac gene, including its signal peptide-encoding sequence,was amplified by PCR using chromosomal DNA from A. utahensisNRRL 12052 as a template (8, 18). The PCR primers were designedaccording to the DNA sequence of aac (8). The primers used wereAAC53 (5'TGCTCTAGAGGAGGTGCCGCCGTGACGTCCTCGTACATGCGCC-3') andAAC31 (5'CCGGAATTCCTCAGCGTCCCCGCTGTGCCAC-3'). The restrictionsites XbaI and EcoRI are shown in italics, the ribosome bindingsite sequence is underlined, and the start codon is shown inbold. PCR amplification was carried out under standard conditionswith a Mastercycler gradient thermocycler (Eppendorf, Germany).Purified PCR products (2.5 kb) were digested with XbaI and EcoRIendonucleases and cloned into the pEM4 vector. The resultingplasmid, pEAAC (Fig. 2), which includes the Streptomyces ribosomebinding site consensus sequence and the putative GTG start codon(8), was introduced in S. lividans 1326 by standard procedures(10). The highest AuAAC production (1.264 U/ml) was reachedafter 96 h of incubation of the recombinant S. lividans (pEAAC)strain. Remarkably, the AuAAC yield was 21-fold higher in therecombinant strain than in A. utahensis (Table 1). More important,a heterodimeric form of homogeneous AuAAC was present in thefermentation broth of S. lividans (pEAAC), in contrast to theincomplete, processed forms of heterogeneous AuAAC describedpreviously (8, 9).

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FIG. 2. Scheme of the recombinant plasmid pEAAC. Numbers indicate the positions of relevant sequences. The length of the plasmid is indicated in base pairs. Abbreviations: apr, ampicillin resistance gene; tsr, thiostrepton resistance gene; pemrE*, ermE* promoter; RBS, ribosome binding site; GTG, start codon; ori pUC, replication origin from pUC plasmid; ori pWHM4, replication origin from pWHM4 plasmid.

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TABLE 1. Production of AuAAC by different microorganisms

Purification and characterization of recombinant AuAAC.

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Purification and...

The recombinant AuAAC produced by S. lividans (pEAAC) was purifiedby two chromatographic steps (Table 2), a procedure considerablyfaster than those previously described which used five purificationsteps (8, 9). Culture supernatants from recombinant S. lividans(pEAAC) cells, grown as described above, were adjusted to pH6.0 and applied onto an S-Sepharose fast-flow column (AmershamBiosciences, United Kingdom) equilibrated with 10 mM sodiumphosphate buffer, pH 6.0. The column was washed with the samebuffer, and bound proteins were eluted with a linear gradientof 0 to 1.5 M NaCl in the same buffer. The fractions showingdeacylase activity were pooled, concentrated with polyethyleneglycol 35000 (reverse dialysis), and loaded onto a Superose12 fast flow column (Amersham Biosciences, United Kingdom).The column was both equilibrated and eluted with 50 mM potassiumphosphate buffer, pH 7.0 (isocratic elution). Routinely, proteinconcentration was determined according to Bradford (5) and thepurity of AuAAC was analyzed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (12).

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TABLE 2. Purification of recombinant AuAAC produced by S. lividans (pEAAC)

Several properties of recombinant AuAAC have been further investigatedby using the purified enzyme. First, the molar extinction coefficientof AAC, Formula , was determined by the method of Edelhoch (7). Second, based on the far-UV circulardichroism (CD) spectrum of AuAAC (Fig. 3), we have determinedthat the protein contains 29% ${alpha}$ -helix, 18% ß-sheet,17% ß-turns, and 36% random coil. Remarkably, CD-thermaldenaturation experiments revealed that the AuAAC global structurewas highly thermostable, showing a midpoint transition temperatureof 81.5°C (Fig. 3, inset).

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FIG. 3. Far-UV CD spectra of pure AuAAC. Spectra were recorded at 0.28 mg/ml in 50 mM potassium buffer, pH 7.0, at 25°C between 175 and 240 nm under thermostated conditions by using a JASCO 715 (Japan) spectropolarimeter. The CD readings were expressed as mean residue molar ellipticities (deg.cm² dmol^–1), assuming a residue molecular weight of 110. (Inset) Thermal unfolding of AuAAC was studied by recording the molar ellipticity variations at 220 nm for temperatures between 25 and 85°C scanned at 20°C/hour. Secondary-structure information was obtained from CD spectra by the CCA (14) and CDNN V2.0.3.188 (4) programs.

Furthermore, the thermal stability of ß-lactam acylaseactivity was studied by preincubating the recombinant AAC for20 min at different temperatures (30 to 80°C). The activitywas stable up to 50°C, slowly decreasing up to 75°Cand becoming drastically reduced at higher temperatures. Theseresults correlate with those for the CD-thermal denaturationstudies, where unfolding was observed at high temperatures.In addition, the effect of temperature on AAC was examined bymeasuring its activity in the temperature range of 30 to 80°C,using penicillin V as a substrate. Surprisingly, the highesthydrolytic activity was achieved at 75°C.

Finally, AAC activity was assayed at different pH levels. Usingpenicillin V as a substrate, we observed that the optimal pHranged between 8.0 and 8.5.

Substrate specificity of recombinant AuAAC.

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Substrate specificity of...

As pointed out above, complementary analyses carried out inour laboratory showed that this enzyme was very similar to thepenicillin V acylase from S. lavendulae, strongly suggestingthat AuAAC might behave as a true ß-lactam acylase.Remarkably, in agreement with this hypothesis, we have determinedthat purified recombinant AuAAC is able to hydrolyze penicillinV as penicillin V acylase does (Fig. 1B), in contrast to theechinochandin B acylase from A. utahensis (an isoenzyme of AuAAC),which is apparently unable to hydrolyze penicillin V (11). Forsuch hydrolysis, the enzyme does not require any external cofactor,metal ion, or reducing agent for maximal activity, althougha slight stimulation of activity was found in the presence of0.6 M KCl (data not shown). This result demonstrated that AuAACcan certainly be reclassified as a ß-lactam acylase,not only according to its primary structure but also based onits enzymatic activity, and therefore, we decided to study indetail its catalytic parameters, using ß-lactam antibioticsas substrates.

To examine the hydrolytic specificity of AuAAC, we determinedthe kinetic parameters for the hydrolyses of different naturalß-lactam antibiotics, i.e., penicillins V, K, F, dihydroF,and G (Table 3). The pure recombinant enzyme (0.5 µg)was incubated with increasing concentrations of the correspondingpenicillins (V, K, F, dihydroF, and G) in 100 mM potassium phosphatebuffer, pH 8.0, at 45°C for 15 min in a final volume of100 µl. The reaction was stopped by addition of 400 µlof 0.5 M sodium acetate. After centrifugation of the samples,the released 6-APA (6-aminopenicillanic acid) was monitoredwith fluorescamine (16, 19). The reaction was linear under theseassay conditions. Values of kinetic constants were determinedby fitting initial velocity data to the Hanes-Woolf equation(17), using a hyperbolic regression program (Hyper.exe 1.01,by J. S. Easterby [http://homepage.ntlworld.com/john.easterby/software.html]).Substrate saturation kinetics were fitted to the equation v= (V_max x S)/(K_m + S).

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TABLE 3. Kinetic parameters of recombinant AuAAC assayed with different natural penicillins

Remarkably, AuAAC shows the same substrate specificity on thesenatural penicillins as penicillin V acylase from S. lavendulae(21). Thus, penicillin K was the best penicillin substrate forAuAAC, showing the highest bimolecular constant (specificityconstant) value, k_cat/K_m = 34.79 mM^–1 s^–1. Thisvalue is within the range reported for its natural substrateaculeacin A (18). It is worth noticing that other ß-lactamacylases of the penicillin G acylase family so far cloned andassayed do not display such hydrolytic capability (1, 2). Likewise,the enzyme hydrolyzes penicillin G at an extremely low rate(2 to 3% of the rate for hydrolysis of penicillin V). Thesestructural preferences for aliphatic chains suggest the existenceof a peculiar hydrophobic pocket in the active center, as describedfor penicillin acylase from S. lavendulae (21).

These results taken together suggest that AAC from A. utahensisand penicillin acylase from S. lavendulae can be consideredthe first members of a new subfamily of ß-lactam acylasesshowing high specificity for penicillin K, and we propose toclassify them as penicillin K acylases. Likewise, AuAAC shouldbe considered an industrial biocatalyst with high potentialin the production of semisynthetic penicillins.

ACKNOWLEDGMENTS

We express our gratitude to J. A. Salas from the Universityof Oviedo for providing the pEM4 expression vector.

This work was supported by grant BIO 2003-04832 from the SpanishMinistry of Education and Science.

FOOTNOTES

* Corresponding author. Mailing address: Departamento de Bioquímica y Biología Molecular I Facultad de Biología, Universidad Complutense, C/ José Antonio Nováis 2, 28040 Madrid, Spain. Phone: 34-91-3945120. Fax: 34-91-3944672. E-mail: isabel{at}bbm1.ucm.es

Published ahead of print on 22 June 2007.

REFERENCES

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