Penicillin acylases revisited: importance beyond their ...

http://informahealthcare.com/bty ISSN: 0738-8551 (print), 1549-7801 (electronic) Crit Rev Biotechnol, Early Online: 1–14 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/07388551.2014.960359

REVIEW ARTICLE

Penicillin acylases revisited: importance beyond their industrial utility Vellore Sunder Avinash, Archana Vishnu Pundle, Sureshkumar Ramasamy, and Cheravakkattu Gopalan Suresh

Critical Reviews in Biotechnology Downloaded from informahealthcare.com by National Chemical Laboratory on 11/28/14 For personal use only.

Division of Biochemical Sciences, CSIR-National, National Chemical Laboratory, Pune, India

Abstract

Keywords

It is of great importance to study the physiological roles of enzymes in nature; however, in some cases, it is not easily apparent. Penicillin acylases are pharmaceutically important enzymes that cleave the acyl side chains of penicillins, thus paving the way for production of newer semi-synthetic antibiotics. They are classified according to the type of penicillin (G or V) that they preferentially hydrolyze. Penicillin acylases are also used in the resolution of racemic mixtures and peptide synthesis. However, it is rather unfortunate that the focus on the use of penicillin acylases for industrial applications has stolen the spotlight from the study of the importance of these enzymes in natural metabolism. The penicillin acylases, so far characterized from different organisms, show differences in their structural nature and substrate spectrum. These enzymes are also closely related to the bacterial signalling phenomenon, quorum sensing, as detailed in this review. This review details studies on biochemical and structural characteristics of recently discovered penicillin acylases. We also attempt to organize the available insights into the possible in vivo role of penicillin acylases and related enzymes and emphasize the need to refocus research efforts in this direction.

b-lactam, choloyl glycine, in vivo role, Ntn hydrolases, pathogenicity, quorum sensing, regulation, signalling

Introduction Penicillin acylases (PAs, E.C. 3.5.1.11) have been long studied as enzymes important in the pharmaceutical industry. They catalyze the deacylation of natural penicillins to the active pharmaceutical intermediate 6-aminopenicillanic acid (6-APA). Together, penicillin acylases and cephalosporin acylases (that deacylate cephalosporins to 7-deacetoxy (7-ADCA) or 7-amino (7-ACA) cephalosporanic acid) are now widely used in the production of semisynthetic antibiotics (Shewale & Sivaraman, 1989). Penicillin acylases are classified based on their substrate specificity (Deshpande et al., 1994), into penicillin G acylases (PGAs, preferentially hydrolyzing benzylpenicillin or pen G) and penicillin V acylases (PVAs, preferentially hydrolyzing phenoxymethyl penicillin or pen V). Although penicillin acylases have been characterized from a diverse range of bacteria and fungi (Arroyo et al., 2003; Shewale & Sudhakaran, 1997), the Escherichia coli PGA (EcPGA) has been widely studied and used in the industry owing to the ease of operation and economics (Shewale & Sivaraman, 1989). Besides the production of semi-synthetic antibiotics, penicillin acylases are also employed in peptide synthesis and in the resolution of racemic mixtures (Arroyo et al., 2003).

Address for correspondence: Sureshkumar Ramasamy and C.G. Suresh, CSIR-National Chemical Laboratory (CSIR-NCL), Pune 411008, India. Tel: 020-25902236. E-mail: [email protected]; cg.suresh@ ncl.res.in

History Received 28 March 2014 Revised 15 July 2014 Accepted 29 July 2014 Published online 26 November 2014

Many authors have reviewed the use of penicillin acylases in the pharmaceutical industry, and the methods used to improve the process economics, including mutagenesis, protein engineering, enzyme production and immobilization and advancements during downstream processing (Arroyo et al., 2003; Chandel et al., 2008; Grulich, 2013; Parmar et al., 2000; Shewale & Sudhakaran, 1997; Sio & Quax, 2004). However, the importance of these enzymes in the industrial arena has overshadowed the preliminary efforts to study their biological role in the natural metabolism of the microorganisms that produce them. Earlier studies have been limited to studying the effect of environmental parameters that regulate enzyme production or inhibition (Vandamme & Voets, 1974; Valle et al., 1991). The observed catabolite repression of these enzymes by glucose has led to a hypothesis that penicillin acylases could function as scavenger enzymes (Valle et al., 1991). Although, the pac gene of E. coli encoding the PGA enzyme has been found to be associated with the phenylacetic acid (PAA) catabolic operon (Galan et al., 2004), there are no such studies on PVAs; these are structurally closer to conjugated bile acid hydrolases (CBAHs; Kumar et al., 2006). Over the last decade, there have been some reports on the role of penicillin acylases in microbial metabolism. Also, enzymes that bear a structural and sequence similarity to penicillin acylases have also been structurally characterized. This review attempts to organize the insights obtained so far on the biological role of penicillin acylases in nature, and emphasizes the need for further exploration in this direction.

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V. S. Avinash et al.


Biochemical and structural characteristics of penicillin acylases The advent of widespread antibiotic resistance among bacteria and other microorganisms has led to the characterization and development of b-lactam modifying enzymes (acylases) for the production of semi-synthetic antibiotics. Several of these enzymes, identified in the 1960s and 1970s, were classified as penicillin or cephalosporin acylases based on their ability to produce 6-APA or 7-ACA (Rolinson, 1988). Our understanding of these enzymes and their related homologues has improved vastly over the years. It is now known that b-lactam acylases act on a wide range of substrates and show cross-reactivity, and that they could act on completely different substrates in nature. Thus, although the focus of the review is on penicillin acylases, the argument for studying their role in natural physiology could be considered equally significant in the case of cephalosporin acylases, other homologues and b-lactam acylases in general. Penicillin acylases have been isolated and characterized from a multitude of bacteria and fungi (Rajendhran & Gunasekaran, 2004; Shewale & Sudhakaran, 1997). Previously, PGAs were classified as bacterial acylase, while PVAs were considered as fungal acylases (Vandamme & Voets, 1974). However, now it is known that many bacteria (Shewale & Sudhakaran, 1997) produce PVAs also. PGAs from E. coli (Duggleby et al., 1995), Alcaligenes faecalis (Verhaert et al., 1997; Varshney et al., 2012), Providencia rettgeri (Klei et al., 1995; McDonough et al., 1999) and Kluyvera citrophila (Kim et al., 2004a,b; Varshney et al., 2013) have been well-studied with respect to their structure and post-translational processing. While these enzymes are targeted at the periplasmic space, the PGAs from Grampositive bacteria Bacillus megaterium (Senthilvel & Pai, 1998; Yang et al., 2006) and Arthrobacter viscosus are extracellular (Konstantinovic et al., 1994; Ohashi et al., 1989). Intracellular PGAs have been characterized from Bacillus badius (Rajendhran & Gunasekaran, 2007); and Achromobacter sp. (Skrob et al., 2003) which shows higher activity towards ampicillin. An enzyme PAS2, with improved synthetic properties, has been isolated from sand soil enrichment culture (Gabor et al., 2005). PVAs studied so far from Gram-positive bacteria, such as B. sphaericus (Olsson et al., 1985) and B. subtilis (Rathinaswamy et al., 2005), are cytoplasmic, while the pva genes from Gramnegative bacteria have a periplasmic signal sequence (Kovacikova et al., 2003). However, PVAs from fungi, including Penicillium chrysogenum (Erickson & Bennett, 1965), Pleurotus ostraetus (Stoppok et al., 1981) and Fusarium sp. (Sudhakaran & Shewale, 1995) have been found both intra- and extra-cellularly localized. Thus, there appears to be a prominent diversity of penicillin acylases, even in their source and localization. Tables 1 and 2 provide a detailed overview of the characteristics of penicillin acylases and other structurally related enzymes described later in this review. With regard to their three-dimensional structure and catalytic mechanism, penicillin acylases belong to the Ntn hydrolase family, which are characterized by a catalytic N-terminal nucleophile residue and a abba-fold (Brannigan

Crit Rev Biotechnol, Early Online: 1–14

et al., 1995; Oinonen & Rouvinen, 2000; Suresh et al., 1999). Besides penicillin acylases, this family also includes glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferases (GRPP), proteasome and aspartyl glucosaminidase. The catalytic mechanism of Ntn hydrolases involves a nucleophilic attack by the N-terminal residue on the substrate carbonyl carbon of the amide bond; the base accepting the proton from the nucleophile is the free N-terminal a-amino group of nucleophile residue (Brannigan et al., 1995; Oinonen & Rouvinen, 2000). Despite acting on structurally related substrates, PGA and PVA have been observed to be different in their biochemical and structural characteristics, including the N-terminal nucleophile (PGA-ser, PVA-cys; Oinonen & Rouvinen, 2000). PGAs are usually heterodimeric, while the PVA from B. sphaericus forms a homotetramer (Arroyo et al., 2003; Suresh et al., 1999). Processed PGAs have a molecular weight in the range of 20–24 kDa for the a-subunit and 60–65 kDa for the b-subunit (Rajendhran & Gunasekaran, 2004), while the size of PVA monomer varies from 30 to 35 kDa (Shewale & Sudhakaran, 1997). Penicillin acylases undergo post-translational autoproteolytic processing to produce the active form of the enzyme, although the processing steps differ between PGA and PVA (Duggleby et al., 1995; Suresh et al., 1999). The pac gene of EcPGA has an N-terminal signal sequence that directs the protein to the periplasm, and the a-and b-subunits are separated by a spacer peptide or pro-peptide (Schumacher et al., 1986). The propeptide is processed by intramolecular proteolysis to yield a mature two-chain protein (Figure 1; Brannigan et al., 1995; Sudhakaran et al., 1992). The N-terminal serine of the b-subunit acts as the catalytic nucleophile residue. The structure of PGAs from E. coli and P. rettgeri mutants, which have impaired processing, reveal tightly bound calcium (Ca2+) ion in the protein. The calcium ion has been shown to coordinate two phenyl alanine residues (aPhe71 and bPhe 146), thus joining the two chains and defining the structure of the active site (Duggleby et al., 1995; McVey et al., 2001).

Post-translational processing of PAs Kasche et al., (2003) have studied the folding and maturation of PGA from Alcaligenes faecalis (AfPGA), which shows 40% sequence similarity to EcPGA and a higher specific activity. AfPGA has a striking difference in the length of the propeptide that is processed. Addition of fragments of the internal pro-peptide activated the mature enzyme with a 2.3-fold increase in specific activity; and processing mutants containing attached regions of pro-peptide to the a-chain showed an increased kcat value while maintaining a similar Km. The authors have hypothesized that the pro-peptide influences the kinetic constants of the PGA enzyme possibly by stabilizing a transition state during the reaction (Kasche et al., 2003; Ignatova et al., 2005). Subtle differences exist across molecules in the processing pathway and catalytic machinery, even within PGAs. There are reports that AfPGA is transported to the periplasm via the Sec-translocation pathway (Kasche et al., 2005) while EcPGA is directed to the Tat-pathway with a modified signal peptide (Ignatova et al., 2002). In contrast, the

– Km ¼ 0.35 mM Km ¼ 0.67 mM

C8, C10, oxoC12 AHL, Pen G glutaryl 7-ACA glutaryl 7-ACA glutaryl 7-ADCA

Pseudomonas sp. C427 Brevundimonas diminuta

–

–

Streptomyces sp. (AhlM)

Ralstonia sp.

– 7.8 104 0.14 104

C7-14 AHLs C8 AHL 3-oxo C12 AHL Pen V, G oxo C8-12 AHL

2.08 106 6 106 –

Km ¼ 1.83 mM 1.025 106 0.805 106 2.1 104 8.6 104 5.82 104 8.2 106 2.6 106 1.5 107 4.2 106

periplasmic

Periplasmic, membraneanchored extracellular

Periplasmic

Used in synthesis of antibiotics

–

–

Isolated from environmental gene pool Hydrophobic binding pocket to accommodate long chain AHLs – –

2x active on ampicillin and cephalexin than pen G

Cytoplasmic

Periplasmic

thermostable

– Broad substrate specificity

Periplasmic

Intracellular

–

Pyoverdine biosynthesis, not required for AHL utilization AHL utilization and recycling, cell signalling turnover

Kim et al. (2001)

Nagao et al. (2004)

Park et al. (2005)

Lin et al. (2003)

Sio et al. (2006), Bokhove et al. (2010), Jimenez et al. (2010) Huang et al. (2006) Wahjudi et al. (2011)

Gabor et al. (2005)

Skrob et al. (2003)

Cai et al. (2004)

De Souza et al. (2005) Rajendhran et al. (2007)

Ohashi et al. (1988)

–

Km ¼ 0.42 mM

Roa et al. (1994), Mukherji et al. (2014)

Sevo et al. (2002)

Extracellular

References Margolin et al. (1980)

–

Possible scavenging of phenylacetyl compounds, cell signalling

Metabolic pathways involved

Svedas et al. (1997)

Enantioselectivity, resolution of racemic mixtures, antibiotic and peptide synthesis –

Special characteristics and applications

Thermostable, enantioselective

Periplasmic

Cellular location

3.1 106 2.5 106 0.651 103 0.103 103 1.3 107 2.1 107 Km ¼ 19.7 mM

1 107 1.7 106

Kcat/Km values (M1s1)

P. aeruginosa (quiP) P. aeruginosa (PA0305)

P. aeruginosa (PvdQ)

PAS2

Achromobacter xylosoxidans Achromobacter sp.

Arthrobacter viscosus B. megaterium B. badius

Providencia rettgeri

Pen G Pen G NIPAB ampicillin cephalothin cephalexin Pen G NIPAB Pen G NIPAB Ampicillin cephalexin Pen G NIPAB C7-14 AHLs

Pen G NIPAB C6-AHL oxo C6-AHL Pen G NIPAB NIPAB Pen G Pen G

K. citrophila

A. faecalis

Pen G NIPAB

Substrates hydrolyzed

E. coli

Source organism

AHL, Acyl homoserine lactone; NIPAB, 5-nitro 2-(phenylacetamido) benzoic acid; ACA, amino cephalosporanic acid

Cephalosporin acylase

AHL acylase

Penicillin G acylase

Enzyme

Table 1. Characteristics of penicillin G acylases and related enzymes from different bacteria.


DOI: 10.3109/07388551.2014.960359

Biological importance of penicillin acylases 3

Thermus thermophilus

S. mobaraensis

Streptomyces lavendulae

Pen K Pen F Pen V Pen V NIPOAB Capsaicin Pen G Pen K Pen F Pen G

GDCA Bile salts Bile salts

Brevibacillus sp. Listeria monocytogenes Brucella abortus

Bifidobacterium longum

R. aurantiaca Clostridium perfringens

Lactobacillus plantarum

Pen V GDCA Pen V Pen V

Substrates hydrolyzed

Pen V Pen V, cloxacillin, tripeptides Pen V GCA GDCA GCDCA TCA TDCA TCDCA Pen V GCA TCA Bile salts

Fusarium oxysporum E. aroideae

B. subtilis Aeromonas sp. ACY95

B. sphaericus

Source organism

Extracellular

1.65 105 2.19 104 3.88 104 5.19 104 6.65 104 1.14 105 2.1 103 1.61 104 0.96 103 0.77 103 Membrane anchored

Cytoplasm Intracellular

Cytoplasm

Cytoplasm

Cytoplasm

Intracellular

Cytoplasm

Cellular location

Km ¼ 3 mM – –

3.8 106 2.4 106 –

0.24 103 Km ¼ 3.6 mM 1.2 mM 14 mM 37 mM 3.5 mM 3 mM

Km ¼ 5.2 mM Km ¼ 31 mM

0.3 104 –

4.1 10

3

Kcat/Km values (M1s1)

Thermophile origin

Isolated as capsaicin hydrolase

Pen K acylase

Specificity for bile salts, no PVA activity 4 BSH proteins, bsh1 shows preference for GDCA Thermophilic BSH – –

monomeric PVA Affinity for deoxy bile conjugates

Specific for pen V Production induced by phenoxyacetic acid Production induced by POAA Constitutive production

Hydrolyzes bile salts to 20%

Special characteristics and applications

Pathogenesis, bile resistance, colonization and establishment of infection Unknown, possibly degradation of long chain fatty acid amides

Bile tolerance, cholesterol reduction

Unknown, possibly scavenging for alternative carbon sources

Metabolic pathways involved

Torres et al. (2012)

Zhang et al. (2007)

Lambert et al. (2008a), Ren et al. (2011) Sridevi et al. (2009) Dussurget et al. (2002) Delpino et al. (2007, Marchesini et al., 2011) Torres Guzman et al. (2002)

Kumar et al. (2006)

Kumar et al. (2008) Nair et al. (1967), Rossocha et al. (2005)

Lowe et al. (1986) Vandamme & Voets (1975)

Olsson et al. (1985), Suresh et al. (1999) Rathinaswamy et al. (2005) Deshpande et al. (1996)

References


GDCA, glycodeoxycholic acid; GCA, glycol cholic acid; GCDCA, glycochenodeoxycholic acid; TCA, taurocholic acid; TDCA, tauro deoxy cholic acid; TCDCA, tauro chenodeoxy cholic acid; POAA, phenoxy acetic acid; NIPOAB, 5-nitro 2-(phenoxyacetamido) benzoic acid.

Aliphatic penicillin acylases

Bile salt hydrolase

Penicillin V acylase

Enzyme

Table 2. Characteristics of penicillin V acylases and related enzymes.


4 Crit Rev Biotechnol, Early Online: 1–14


DOI: 10.3109/07388551.2014.960359

Biological importance of penicillin acylases

5

Figure 1. Structure of (a) processed (Protein Data Bank Code 1PNK) and (b) slow-processing mutant precursor (PDB code 1E3A) of EcPGA. (The slow-processing mutant precursor retains the spacer peptide). The important residues have been highlighted.

processing mechanism of PVAs is simpler, with the methionine at the start of the sequence (in Gram-positive bacteria) cleaved autocatalytically to reveal the N-terminal cysteine. The PVA from B. sphaericus (BspPVA) has been reported (Suresh et al., 1999) to process a tripeptide (Met-Leu-Gly) at the N-terminal end of the catalytic cysteine, while the B. subtilis PVA (Rathinaswamy et al., 2005) contains only a methionine before the N-terminal residue. Site-directed mutational studies (Chandra et al., 2005) have shown that the BspPVA needs a conserved Asn175 as oxyanion hole residue to stabilize the transition state complex during processing and catalysis; while Arg17 acts as base to deprotonate the Sc of cysteine and Asp20 stabilizes the protonated a-amino group. Although B. sphaericus employs an intramolecular autoproteolytic mechanism for activation of the enzyme, in general the removal of the starting methionine seems to occur without the involvement of the N-terminal cysteine. Based on the sequence of the pva gene in V. cholerae, Kovacikova et al. (2003) have proposed that Gram-negative PVAs undergo processing by cleavage of the periplasmic signal peptide. However, it is not yet known through which secretion pathway the PVAs are processed.

Catalytic mechanism and structural organization Both PGAs and PVAs have been shown to share, with cephalosporin acylases, similar catalytic residues that spatially overlap (Figure 2), and an identical structural fold that helps them cleave chemically related substrates and places them in the same superfamily (Suresh et al., 1999). The catalytic reaction mechanism of PGAs involves a nucleophilic attack by the N-terminal serine (bSer1) on the carbonyl carbon of the amide bond of the substrate benzylpenicillin; and the formation of a tetrahedral intermediate stabilized by oxyanion hole residues helps in release of the product (McVey et al., 2001). A more recent study, based on quantum mechanical models (Zhiryakova et al., 2009), proposes that

bGln23 and bAsn241 residues are also involved in the complex interaction, beside bSer1. It has also been shown that a pair of arginine residues (bArg145and aArg263) play a crucial role in the substrate binding and influence the stereoselectivity of PGA enzyme to a certain extent (Guncheva et al., 2004). The catalytic mechanism of PVAs and the closely related bile salt hydrolases (BSHs) (Kumar et al., 2006) is a little different compared to PGAs, with Cys1 acting as the nucleophile, favoring direct proton transfer from the bridging water molecule as shown in Figure 3 (Suresh et al., 1999). The amino acid residue Asp 274 has been shown to interact with the extra oxygen in pen V, possibly enabling substrate recognition over pen G (Chandra et al., 2005). A quantum mechanics simulation-based study (Lodola et al., 2012) offers new insights into this mechanism. The authors have suggested a reaction path involving a chair-like transition state complex, stabilized by various conserved residues in the catalytic site including Asn82 and Arg 228. In addition, the N-terminal cysteine has been shown to participate in catalysis in a zwitter ionic form (S/NHþ 3 ion pair). The secondary structure arrangement of these two enzymes seems to imply divergent evolution from a common ancestor. Nevertheless, these enzymes are distant enough to suggest that they might be involved in different roles in nature (Suresh et al., 1999). This claim is also substantiated by evidence on the specificity of the enzymes for the acyl chain and b-lactam group in penicillins. PGA shows less specificity for the betalactam nuclei, and a variety of substituents can be accepted. Hence, it can hydrolyze different compounds attached to the phenylacetyl group through an amide bond. It has also been shown to act on substrates with an N-acetyl group (Cole, 1969).This broad spectrum activity of PGA has been exploited in the resolution of racemic mixtures (Cardillo et al., 1996; Ismail et al., 2008; van Langen et al., 2000), as the enzyme specifically cleaves the amide bond of only the L-isomer of the phenylacylated derivative (van Langen et al., 2000). Mutant


6



(a)

(b)

PVA Cys 1 Asp 20 Tyr 82 Asn175 Arg 228

PGA Ser B1 Gln B23 Ala B69 AsnB241 Arg B263

Gl-7ACA Ser B1 His B23 Val B70 AsnB244 Arg B274

Figure 2. Comparison of the active sites of PVA (stick representation) with PGA (a) and Glutaryl 7-ACA acylase (Gl-7ACA) (b). Some of the important residues in the active sites of these enzymes (inset) are shown spatially overlapping.

forms of cephalosporin acylase have also been reported to accept amino acids in place of the b-lactam group (Sio et al., 2002). On the other hand, PVAs have been observed to require the intact b-lactam ring for activity, and are specific for phenoxymethyl penicillin as a whole unit. The Erwinia aroideae PVA has been reported to have no activity on penicilloic acids, or on N-acetyl amino acids (Vandamme & Voets, 1975).

Physiological roles of penicillin acylases Although PGAs and PVAs work on similar penicillin derivatives and structurally belong to the same family (Ntn hydrolases), they show remarkable differences in their oligomeric nature, processing and substrate specificity. In addition, it is probable that these enzymes work on different substrates other than penicillin in natural habitats, since the ability to deacylate penicillin cannot be considered as conferring evolutionary advantage (Cole & Sutherland, 1966; Valle et al., 1991). Suresh et al. (1999) have explained the structural similarity of PGA and PVA in spite of the absence of significant sequence homology. As evident from such structural studies, it is of great interest and importance to study the natural role and evolution of these Ntn hydrolases, especially penicillin acylases. Unfortunately, due to the industrial importance of these enzymes, the overwhelming focus of the studies had been on the application properties rather than on the biological function. There have been some related studies to understand the biological role of these enzymes, which have provided some pointers to the probable roles of penicillin acylases in nature and their evolution, as discussed below.

PGA in the degradation of phenylacetyl compounds Hypotheses on the role of PGAs in nature indicate their function as a scavenger enzyme that acts on alternative carbon sources, compounds that have a phenylacetyl group (Valle et al., 1991). The native E. coli enzyme is repressed by glucose acting at the transcriptional level while production is induced by PAA (Merino et al., 1992), furthering this hypothesis. In E. coli, the pac gene coding for penicillin acylase has been found to be related to the hydroxyphenylacetic acid catabolic operon (Prieto et al., 1996). Kim et al. (2004a,b) have demonstrated that the gene for penicillin acylase in E. coli is regulated by the binding of a specific PaaX repressor to the pac promoter. The paaX regulator controls the expression of the phenylacetyl coA catabolon, which is located distant along the sequence from the pac gene. This process implicates the probable involvement of the PGA enzyme in the metabolism of phenylacetyl-coA (PA-coA)-related compounds. When PAA is present, it is converted into PA-coA by a specific ligase (PaaK), which prevents the PaaX repressor from binding and thus relieves the repression on the synthesis of PGA. In fact, the induction of PGA production by PAA in the medium seems to be achieved through the derepression of the pac gene by PA-coA. As shown by Galan et al. (2004) and Kim et al. (2004a,b), the E. coli paa mutant showed a constitutive high expression and synthesis of PGA. Also, the pac promoter has been observed to be a CRP (cAMP repressor protein)-dependent promoter, leading to the conclusion that the cAMP-CRP complex is essential for the activation of pac expression. This could explain the repression of PGA production in the presence of glucose in the medium (Kim et al., 2004a,b; Valle et al., 1986).


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Arg 17 Cys 2

2

Tyr 82


NH2

O

Asn 175

SH NH2+

6

R'

H NH

Tyr 82

NH3+

Asp 21 O−

NH3+

NH2

Tyr 82 O

Asn 175

R'

HO

O−

O

Arg 228

O

Asn 175

H

R' O

NH

Arg 17

NH2+

5

O

NH3+

Arg 228 O

NH3+

S

H

Arg 228

Tyr 82

Asn 175

NH2

Cys 2

Asp 21 OH

NH2+

H

NH3+

Cys 2

NH3+

S

NH

Arg 17

Arg 17

Cys 2

R' O

NH2

O

NH2

R

O-

Arg 228

Asn 175

O

H

R

NH3+

NH

NH2

Arg 228

H

3

H N

O

NH3+

NH2+ H

R'

R'

R

Asp 21

O−

NH2+

NH3+

S

H

N

1

Asp 21

NH3+

S OH

H

4

Asp 21

NH2+ H

O−

Cys 2

SH

O−

Arg 17

Arg 17 Cys 2

NH3+

Asp 21

7

NH

Arg 228 NH3+

NH2

NH2

Tyr 82

Tyr 82 O

Asn 175

Figure 3. Reaction mechanism of penicillin acylases (amino acids based on B. sphaericus PVA). The sulfhydryl group of the N-terminal cysteine (stabilized by Arg18) performs a nucleophilic attack (1) on the carbon atom of the amide bond of penicillin and related substrates. The tetrahedral intermediate is stabilized by the oxyanion hole (2), involving Asn175 and Tyr82. After the release of the leaving amino product (3), the second nucleophilic attack is carried out by a protonated water molecule on the thioester bond between Cys2 and the substrate. A similar tetrahedral intermediate is formed (5), eventually leading to the liberation of carboxylic acid and regeneration of free enzyme (6).

Galan et al. (2004) have also studied the presence of paaX regulator homologues in other PGA producing strains. K. citrophila, B. megaterium and P. rettgeri were found to lack a paaX homologue, explaining the constitutive nature of PGA production in these bacteria. However, the K. citrophila gene still retained a paaX recognition sequence, which led to repression of PGA synthesis when the gene was expressed in E. coli K12 strain. The presence of a gene coding for PGA even when the bacteria does not have the capability to metabolize PA-coA led the authors to hypothesize that penicillin acylase might be involved in other unknown roles in nature. Apart from such evidence, the recent discovery of novel enzymes structurally similar to PGAs and involved in bacterial cell signaling (discussed below) also provides convincing arguments to explore the alternative physiological roles of PGAs in nature.

Acyl homoserine lactone (AHL) acylases involved in signaling are homologous to PAs In recent years, the bacterial cell signaling phenomenon, quorum sensing (Fuqua et al., 2001), has been the subject of widespread interest. Quorum sensing allows bacteria to

coordinate gene expression and communication with other cells and the environment. The phenomenon is based on the detection of a critical cell density achieved through sensing the concentration of small signaling molecules produced and released by the bacteria (Miller & Bassler, 2001). Accumulation of a minimal threshold concentration of the signal causes alterations in gene expression, thus regulating varied physiological activities. The silencing of quorum sensing (Dong et al., 2001) allows for control of bacterial virulence and other infection-related phenomena. AHL acylases (Leadbetter & Greenberg, 2000) are a group of enzymes involved in such signaling disruption and these enzymes have been found to possess significant sequence and structural similarity with b-lactam acylases and Ntn hydrolases (Sio et al., 2006). AHL acylases act on the acyl chain of AHLs, one of the primary signalling molecules in bacterial quorum sensing. AHLs consist of a homoserine lactone moiety and an acyl fatty acid chain, the length and substitution of which can vary among different bacteria (Miller & Bassler, 2001). Quorum sensing in many Gram-negative bacteria depends on the synthesis and secretion of AHLs, which at a critical concentration bind to specific receptors, ultimately regulating the


8


expression of virulence and other gene systems (Fuqua et al., 2001). AHL acylases have been characterized so far from Pseudomonas aeruginosa (Sio et al., 2006), Rhodococcus erythropolis (Uroz et al., 2005), Ralstonia sp. XJ12B (Lin et al., 2003) and Streptomyces sp. M664 (Park et al., 2005). As already mentioned, these enzymes show significant sequence homology with b-lactam acylases including PGA and cephalosporin acylase (Matsuda et al., 1987) and Aculeacin A acylase (Inokoshi et al., 1992). P. aeruginosa PAO1 has been reported to produce at least four different AHL acylases (Krzeslak et al., 2007). Bokhove et al., (2010) have elucidated the structure of an AHL acylase (PvdQ) from P. aeruginosa, which bears a striking resemblance to PGA structures and shares 24% sequence identity with Ec PGA. It has a abba structural fold typical of Ntn hydrolases and is synthesized as a precursor that forms a heterodimeric protein by autocatalytic processing. Though PvdQ also has serine as N-terminal nucleophile, it differs from PGA in having three disulfide bonds which are conserved in aculaecin acylase and PVA from Streptomyces mobaraensis. The PvdQ catalytic mechanism has been explained to be similar to that of PGA, with the activation of bSer1 by water and the formation of an oxyanion transition intermediate. However, it was also observed that PvdQ contains a hydrophobic pocket near the N-terminal nucleophile in the interior of the enzyme, closed by bPhe24 acting as a gate. This pocket undergoes a volume increase as a result of conformational changes upon binding of the substrate and this induced fit probably helps the enzyme to catalyze long chain C12-HSLs. The secondary structure elements involved in formation of the substrate-binding pocket of PvdQ are similar as found in PGA and cephalosporin acylase. However, small changes in conformation of amino acid side chains in the active site and the replacement of key substrate-binding residues with smaller side chain amino acids makes PvdQ capable of binding long chain AHLs. The aculaecin acylase from Actinoplanes utahensis and PVA from Streptomyces mobaraensis (discussed later) have a similar substrate spectrum and active site organization, thus possibly forming a group of Ntn hydrolases that hydrolyze long chain acyl amides. In addition, PvdQ is probably involved in pyoverdin biosynthesis and its expression is enhanced in conditions of limited availability of iron (Jimenez et al., 2010); thus modulating iron uptake and biofilm formation. Koch et al. (2010) have reported that the role of PvdQ in pyoverdine synthesis is conserved among Pseudomonas spp., while its role in quorum sensing seems exclusive to P. aeruginosa. There are reports about cross-reactivity between cell signaling enzymes and penicillin acylases. AhlM from Streptomyces sp. was found to be capable of degrading penicillin G (Park et al., 2005). The AHL-acylase PA0305 from P. aeruginosa shows a low level of activity on penicillin G and V (Wahjudi et al., 2011). A more recent study (Mukherji et al., 2014) showed that the K. citrophila PGA degrades C6-C8 AHLs, although at a much lesser rate than AHL acylases. Ec PGA did not show any activity towards AHLs, despite having a high sequence homology with the K. citrophila PGA. The differences in the active site organization leading to such a broad and varied substrate spectrum


increases the need for extensive research on these Ntn hydrolases, and penicillin acylases in particular, to understand their place in the physiological network process.

Regulation of PVA gene expression As mentioned previously, PVAs are vastly different from PGAs as they possess no appreciable sequence homology, although they contain the same structural elements and show related catalytic mechanisms (Suresh et al., 1999). The metabolic regulation of enzyme production also follows contrasting patterns in these enzymes. While PGA production is induced by the addition of PAA to the medium (Merino et al., 1992), PVAs are mostly constitutive (Shewale & Sudhakaran, 1997; Vandamme & Voets, 1974), with only a few organisms in which the enzyme production is induced by phenoxyacetic acid (POAA), including Aeromonas sp. (Deshpande et al., 1996) and Fusarium oxysporum (Lowe et al., 1986). As in the case of PGAs, there are many reports about repression of PVA enzyme synthesis by glucose (Shewale & Sudhakaran, 1997); although a few organisms like B. sphaericus (Carlsen & Emborg, 1981) are not subject to glucose repression. In Beijerinckia indica, the PVA production is enhanced by the use of sodium glutamate in the medium (Ambedkar et al., 1991). Kovacikova et al. (2003) have reported an indirect link between quorum sensing and PVA expression in Vibrio cholerae, although there is no close homology between PVAs and AHL acylase-related enzymes. In certain strains of V. cholerae, the quorum sensing system influences the expression of the virulence genes through the activity of the LuxO/HapR response regulators. Activation of the tcpPH promoter on the Vibrio pathogenicity island by regulators AphA and AphB initiates the V. cholerae virulence cascade, and is regulated by quorum sensing through the repressive action of HapR on aphA expression. Apart from the tcpPH promoter, AphA was also found to bind efficiently to another region on the small chromosome upstream of a pva gene. This results in the negative regulation of pva expression in response to cell density. Mutagenesis studies have showed that pva is repressed at low cell density when AphA levels are high and derepressed at high cell density when AphA levels are reduced. The fact that the regulation of pva expression and that of virulence genes expression were mutually exclusive, and thus not observed to play any role in virulence, reiterates the hypothesis that penicillin acylases might function as scavenging enzymes in non-parasitic environments. It is also suggested that though the regulation of PVA might occur by other mechanisms, strains that have functional quorum sensing systems regulating PVA expression would have an advantage in certain environments.

Similarity of PVAs to bile salt hydrolases Bacterial PVAs have significant sequence and structural similarity with BSHs; together they make up the choloylglycine hydrolase group (Kumar et al., 2006). Though these enzymes act on the same non-protein amide chemical bond, the substrates are quite different. The steroid moiety of bile salts occupies more space than the corresponding group of pen V (Lambert et al., 2008a,b). Both the enzymes are



DOI: 10.3109/07388551.2014.960359

Loop BSH 1 21-27 2 58-65 5 129-139 8 259-271

9

PVA 21-27 59-66 131-141 262-274

Figure 4. Overlap of the 8 loops surrounding the catalytic site in BlBSH (Bifidobacterium longum) and BsuPVA (Bacillus subtilis). The front view and side view are shown. Four loops 1, 2, 5 and 8 show major differences in length (inset) and play a crucial role in determining the substrate specificity.

usually annotated as CBAH in protein sequence databases (http://pfam.sanger.ac.uk), and it is hard to separate the two enzymes only based on their sequence. In spite of a similar tetrameric structure, subtle changes in the loop structure elements surrounding the active site region show huge changes in catalytic specificity (Figure 4) of these enzymes (Lambert et al., 2008a,b). For instance, PVA from B. subtilis (Rathinaswamy et al., 2012) and P. atrosepticum (Avinash et al., 2013) hydrolyze pen V exclusively, while PVA of B. sphaericus (Kumar et al., 2006; Pundle & Sivaraman, 1997) can hydrolyze bile salts with a specific activity up to 30% of that shown on pen V as the substrate. Similarly, C. perfringens BSH (Rossocha et al., 2005) can hydrolyze pen V to some extent, but the B. longum enzyme (Kumar et al., 2006) exhibits pure BSH activity. Although not many studies have been carried out on PVAs, bile salt hydrolases have been explored more widely due to their potential application in probiotics (Begley et al., 2006). BSHs are mostly found in intestinal microflora and pathogens that colonize the human gut, and act on bile salts including glyco- and tauro- conjugated cholic acids. They probably confer resistance against the anti-microbial activity of bile, thus enabling the persistence of bacteria in the gastrointestinal tract (Bateup et al., 1995; Begley et al., 2005). Bile salt hydrolases have also been reported to play a role in the ability of probiotics to lower cholesterol levels (Klaver & van der Meer, 1993; Taranto et al., 1997). Structures have been elucidated for BSH from Clostridium perferingens (Rossocha et al., 2005) and Bifidobacterium longum (Kumar et al., 2006). It is also noted that (Ren et al., 2011) most BSH occur in Gram positive bacteria (except Bacteroides), while PVAs occur widely in all bacteria. Unlike PVA, the occurrence of multiple BSH homologues in the same bacterium has also

been reported. In L. plantarum, four genes coding BSHs are present (Lambert et al., 2008a,b; Ren et al., 2011). BSHs have also been reported to act as virulence factors involved in pathogenesis. In Listeria monocytogenes (Dussurget et al., 2002), the bsh gene was found to be positively regulated by PrfA, a transcriptional activator for many known Listeria virulence genes. In addition, the loss of the bsh gene led to reduced bacterial infection, colonization and virulence in both intestinal and hepatic phases of listeriosis. The bsh gene also does not have an orthologue in non-pathogenic strains, thus confirming the importance and role of BSH in pathogenesis. The Brucella abortus BSH has also been shown to confer resistance to bile and play an important role in the infectivity of Brucella through the oral route (Delpino et al., 2007). Certain enteric pathogens (E. coli O157:H7; Hamner et al., 2013) are now known to sense bile and use the signal to regulate the location of expression of virulence factors in the host. Begley et al. (2005) have demonstrated that the presence of a bsh and a pva homologue in Listeria monocytogenes, with both enzymes involved in a bile-specific stress response. The pva gene locus is present in only certain strains and has a lower G + C content than the surrounding region, which indicates a recent acquisition. Differences in G + C content as a result of lateral gene transfer have been indicated in pathogenesis and bacterial resistance to stress conditions (Karlin, 1998), and might provide clues to the physiological functions of such genes. Previous studies on Lactobacillus BSH have proposed the horizontal transfer of bsh genes (Elkins et al., 2001), while the Bifidobacterium bifidum bsh gene could be a paralogoue (Kim et al., 2004a,b). In addition, the Lactobacillus salivarius BSH showed higher specificity for tauro-conjugated than glyco-conjugated bile acids

10



(Fang et al., 2009), opposite to the B. longum BSH (Tanaka et al., 2000). Such subtle differences in substrate specificity among bacteria which colonize different locations in the gut indicate the significance of these enzymes in the metabolism of the organism. In addition, bile acids have also been reported to be produced by bacteria in marine environments (Kim et al., 2007), and BSH/PVA homologues are present in many genome sequences of marine bacteria, hinting at the possibility that BSH and PVA might have a more universal role to play than the regulation of virulence in pathogens.

Novel penicillin acylases Over the years, a number of novel penicillin acylases have been characterized from Actinomycetes and Archaebacteria that show more specificity towards aliphatic penicillins such as octanoyl penicillin (pen K). These enzymes show significant differences from known PGAs and PVAs in the arrangement of their active site residues and substrate binding; hence it is probable that penicillin acylases have evolved to perform diverse physiological functions in nature. The extracellular penicillin acylase from Streptomyces lavendulae (Torres-Guzman et al., 2002) is specific towards pen K (kcat/Km ¼ 165), although it hydrolyzes pen V also to a moderate extent (kcat/Km ¼ 39). However, the protein sequence bears no similarity to known bacterial penicillin V acylases. Another penicillin acylase, from Streptomyces mobaraensis (SmPVA, Koreishi et al., 2006), was originally isolated as an acylase that degrades capsaicin (a plant compound that is chemically derived from vanillyl amine) and a spectrum of N-lauroyl-L-amino acids. This acylase was later found to be specific for pen V (Zhang et al., 2007). SmPVA exhibited a heterodimeric subunit composition in contrast to bacterial PVAs, showing greater sequence similarity with SlPVA and AHL acylase PvdQ, with an N-terminal serine in the b-subunit and eight cysteine residues in the protein sequence. Torres et al. (2012) have isolated a penicillin acylase from the archaebacterium Thermus thermophilus that is also more specific for pen K. However, the enzyme could be engineered to hydrolyze pen G to a certain extent by mutagenesis of selected active site residues (Torres et al., 2013). The enzyme was observed to be anchored to the membrane. All these enzymes described above exhibit considerable sequence similarity with the AHL acylase from Streptomyces sp. M0664, P. aeruginosa (PvdQ) and the Aculeacin acylase from Actinoplanes utahensis. The substrate, penicillin K, contains an aliphatic (octanoyl) side chain linked to b-lactam ring; structurally similar to AHLs that have a hydrophobic fatty acid chain linked to the lactone group (Torres et al., 2012; Zhang et al., 2007). Torres-Bacete et al. (2007) have also reported the hydrolysis of pen K by an acylase that hydrolyzes aculeacin, a cyclic lipopeptide. The range of substrates hydrolyzed by penicillin acylases and structurally related enzymes is shown in Figure 5. PGAs only occur in bacteria, but PVAs have been reported to be produced also by fungi and yeast; including Fusarium oxysporum (Lowe et al., 1986), Penicillium sp. (Erickson & Bennett, 1965), Pleurotus ostreatus (Stoppok et al., 1981),


Chainia sp. (Chauhan et al., 1998) and Rhodotorula glutinis (Vandamme & Voets, 1973). In fact, PVA was termed as a ‘‘fungal acylase’’ prior to the characterization of bacterial homologues (Vandamme & Voets, 1974); however, PVAs from fungal sources have rarely been purified and characterized. Kumar et al. (2008) have characterized a monomeric penicillin V-hydrolyzing enzyme from the yeast Rhodotorula aurantiaca, which showed sequence similarity with dehydrogenases and exhibited serine as the N-terminal residue. The multitude of penicillin acylases characterized from a wide variety of organisms, with significant structural and catalytic difference, highlights the gaps in our understanding of the evolution and importance of these enzymes in nature.

Antibiotics in the role of signaling molecules Recently, antibiotics have been hypothesized to act as interspecies signaling molecules at sub-inhibitory concentrations, to regulate homeostasis in microbial communities. Several studies have shown that at low concentrations, antibiotics can transcriptionally modulate a variety of genes (Davies et al., 2006). In P. aeruginosa and E.coli, they can influence the development of antibiotic-resistant biofilms, and trigger the expression of virulence determinants (Hoffman et al., 2005). According to Babic et al., (2010), the aminoglycoside antibiotic tobramycin inhibits the RhlI/R quorum sensing system in P. aeruginosa at a concentration of 25% minimum inhibitory concentration (MIC), thus decreasing the production of the quorum sensing signal C4-HSL (homoserine lactone). Although such studies are rare on b-lactams, it opens the possibility that penicillins, and acylases by extension, could have more significant undiscovered functions in the microbial inter-species signaling network.

The need for a reinvention During a number of decades since the discovery of penicillin, enzymes that can modify b-lactams have been studied extensively to facilitate the continuous production of different antibiotics. Early studies on pencillin acylases were only focused on their industrial potential, including antibiotic production and other applications such as the resolution of racemic mixtures. The penicillin acylase from E. coli is being used widely in various formulations, due to its ability to hydrolyze a wide variety of phenyl acetyl substituted compounds. However, as detailed in this review, penicillin acylases also function in diverse roles in nature, which have not been given much attention. In the last decade, a large volume of research has been conducted on the Ntn hydrolase family of enzymes; including the discovery and structural characterization of different penicillin acylases and homologous enzymes. Nonetheless, the emphasis remained mostly on application rather than elucidating their physiological role, at least in the case of penicillin acylases. Though PGAs and PVAs act on almost similar penicillins, they are entirely different in terms of their oligomeric organization, retaining only the Ntn hydrolase and abba fold, and the key catalytic residues. They also share sequence and structural homology with different groups of enzymes, which hydrolyze other physiologically unrelated substrates. It is possible that these



DOI: 10.3109/07388551.2014.960359

11

Figure 5. Structures of substrates hydrolyzed by penicillin acylases and related enzymes. The CO–NH amide bond is hydrolyzed.

enzymes have separate evolutionary descents, and penicillins may not be their primary substrate. A number of novel penicillin acylases that act on aliphatic penicillins have also been identified, which seem to possess a different active site configuration. Certain penicillin acylases and evolutionarily related enzymes have been implicated in various physiological roles in bacterial cell signaling and pathogenesis. Penicillin acylases also seem to act on a broad range of substrates, showing cross-activity with molecules involved in these metabolic pathways. This is made possible by subtle differences in active site configuration, leading to differences in details of catalysis even in enzymes within the group that show good sequence similarity. The regulation of penicillin acylases occurs through varied mechanisms, including substrate induction, catabolite repression and quorum sensing, although these mechanisms are not universal. In conclusion, penicillin acylases represent an assorted group of enzymes that are produced by various microorganisms, including Fungi, Eubacteria, Actinomycetes and Archaea. Even though these enzymes have been described under one cluster with respect to their industrial utility, there is a large structural and functional diversity existing between these homologues despite the presence of the common Ntn fold. Thus, in spite of their still relevant demand in antibiotic production, there are compelling reasons that the role of penicillin acylases in natural physiology might not be too

small to be discounted. It is thus imperative to adopt a holistic approach to study these enzymes in the metabolic network.

Declaration of interest VSA thanks Council of Scientific and Industrial Research (CSIR), India for the award of Senior Research Fellowship. RSK thanks the Department of Science and Technology (DST), Government of India for Ramanujan Fellowship. The authors report no declarations of interest.

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