Changing the specificity of α-amino acid ester hydrolase toward para-hydroxyl cephalosporins synthesis by site-directed saturation mutagenesis

α-Amino acid ester hydrolases (AEHs) catalyze the synthesis of β-lactam antibiotics containing an α-amino group with decreased activity toward antibiotics with a p-hydroxyl group. The AEH gene from Xanthomonas rubrillineans was cloned and expressed in Escherichia coli. Based on the crystal structure of the AEH and cefprozil complex, 13 residues not directly involved in substrate recognition were mutated individually. The resulting ~1,300 mutants were screened for activity using cefprozil as a model product based on spectrophotometric assay in a 96-well format. Mutants with improved cefprozil synthetic activity revealed the particular importance of positions 87, 131 and 175 for specificity. The mutant V131S with the highest initial rates of synthesis toward three p-hydroxyl cephalosporins showed 23?%, 17?% and 64?% increase in maximum product accumulation of cefadroxil, cefprozil and cefatrizine, respectively.     

The sequence and crystal structure of the alpha-amino acid ester hydrolase from Xanthomonas citri define a new family of beta-lactam antibiotic acylases

alpha-Amino acid ester hydrolases (AEHs) catalyze the hydrolysis and synthesis of esters and amides with an alpha-amino group. As such, they can synthesize beta-lactam antibiotics from acyl compounds and beta-lactam nuclei obtained from the hydrolysis of natural antibiotics. This article describes the gene sequence and the 1.9-A resolution crystal structure of the AEH from Xanthomonas citri. The enzyme consists of an alpha/beta-hydrolase fold domain, a helical cap domain, and a jellyroll beta-domain. Structural homology was observed to the Rhodococcus cocaine esterase, indicating that both enzymes belong to the same class of bacterial hydrolases. Docking of a beta-lactam antibiotic in the active site explains the substrate specificity, specifically the necessity of an alpha-amino group on the substrate, and explains the low specificity toward the beta-lactam nucleus.     

Current state and perspectives of penicillin G acylase-based biocatalyses

In the course of more than 60-year history, penicillin G acylase (PGA) gained a unique position among enzymes used by pharmaceutical industry for production of β-lactam antibiotics. Kinetically controlled enzymatic syntheses of cephalosporins of novel generations in which PGA catalyzes coupling of activated acyl donor with nucleophile belong among the latest large-scale applications. Contrary to rather specific roles of other enzymes involved in β-lactam biocatalyses, the PGA seems to have the greatest potential. On the laboratory scale, other applications with industrial potential were described, e.g., directed evolution of the enzyme to meet specific demands of industrial processes or its modification into the enzyme catalyzing reactions with novel substrates. The fact that β-lactams represent the most important group of antibiotics comprising 65 % of the world antibiotic market explains such a tremendous and continuous interest in this enzyme. Indeed, the annual consumption of PGA has recently been estimated to range from 10 to 30 million tons. The application potential of the enzyme goes beyond the β-lactam biocatalysis due to its enantioselectivity and promiscuity: the PGA can be used for the production of achiral and chiral compounds convenient for the preparation of synthons and active pharmaceutical ingrediences, respectively. These biocatalyses, however, still wait for large-scale application.     

Crystal Structure of Serratia fonticola Sfh-I: Activation of the Nucleophile in Mono-Zinc Metallo-β-Lactamases

Metallo-β-lactamases (MBLs) or class B β-lactamases are zinc-dependent enzymes capable of inactivating almost all classes of β-lactam antibiotics. To date, no MBL inhibitors are available for clinical use. Of the three MBL subclasses, B2 enzymes, unlike those from subclasses B1 and B3, are fully active with one zinc ion bound and possess a narrow spectrum of activity, hydrolyzing carbapenem substrates almost exclusively. These remain the least studied MBLs. Sfh-I, originally identified from the aquatic bacterium Serratia fonticola UTAD54, is a divergent member of this group. Previous B2 MBL structures, available only for the CphA enzyme from Aeromonas hydrophila, all contain small molecules bound in their active sites. In consequence, the mechanism by which these enzymes activate the water nucleophile required for β-lactam hydrolysis remains to be unambiguously established. Here we report crystal structures of Sfh-I as a complex with glycerol and in the unliganded form, revealing for the first time the disposition of water molecules in the B2 MBL active site. Our data indicate that the hydrolytic water molecule is activated by His118 rather than by Asp120 and/or zinc. Consistent with this proposal, we show that the environment of His118 in B2 MBLs is distinct from that of the B1 and B3 enzymes, where this residue acts as a zinc ligand, and offer a structure-based mechanism for β-lactam hydrolysis by these enzymes.     

Two plate-based colorimetric assays for screening α-amino acid ester hydrolase with high synthesis/hydrolysis ratio

α-Amino acid ester hydrolases (AEHs) are enzymes of interest to the semi-synthesis of β-lactam antibiotics with α-amino, such as cephalexin and cefaclor. An undesired side reaction, the hydrolysis of α-amino acid ester, had hindered applications in antibiotics synthesis. Although the enzymes' S/H ratio can be increased by protein engineering, such approaches require a suitable screening assay. Such a screening assay has not yet been described for AEHs. In this paper, we report a 96-well plate format screening procedure for AEHs based on two spectrophotometric assays. To reduce the hydrolysis reaction while maintaining synthesis activity, and to evaluate the effectiveness of the screening strategy, we introduced random mutations in part of the aeh gene from Xanthomonas rubrillineans by error-prone PCR. By a parallel plate-based screening strategy, three mutants with improved S/H ratio, R87L, T132N and N219I, were obtained.     

Improved thermostability of AEH by combining B-FIT analysis and structure-guided consensus method

α-Amino ester hydrolases (AEH, E.C. 3.1.1.43) catalyze the synthesis and hydrolysis of α-amino β-lactam antibiotics. The AEH enzymes have been shown to feature excellent synthetic capability but suffer from poor thermostability. AEH from Xanthomonas campestris exhibits an optimal activity temperature of 25 °C, an observed half-life of 5 min at 30 °C, and a "T-50" value, the temperature at which the half-life is 30 min, of 27 °C. To improve the thermostability of AEH, a modified structure-guided consensus model of seven homologous enzymes was generated along with analysis of the B-values from the available crystal structures of AEH from Xanthomonas citri. A family of stabilized variants was created including a consensus-driven triple variant, A275P/N186D/V622I. Independent NNK saturation of two high B-factor sites, K34 and E143, on the triple variant resulted in our best variant, the quadruple mutant E143H/A275P/N186D/V622I, with a "T-50" value of 34 °C (7 °C improvement) and 1.3-fold activity compared to wild-type.     

Cloning and expression of variants of the glutaryl-7-aminocephalosporic acid acylase of the bacterium <Emphasis Type="Italic">Brevundimonas diminuta</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

The gene coding for glutaryl-7-aminocephalosporic acid acylase (Gl7ACA acylase) of the bacterium Brevundimonas diminuta (BrdGl7ACA), a commercial enzyme widely used in modern biocatalytic technologies for manufacture of β-lactam antibiotics, was cloned. Efficient expression systems for producing a “native” recombinant BrdGl7ACA and its analogs modified by attaching affinity groups—the chitin-binding domain of chitinases A1 and hexahistidine sequence—were designed. It was demonstrated that both the recombinant hybrid proteins and the native Gl7ACA acylase produced in E. coli cells underwent a correct autoproteolytic processing with generation of functionally active enzymes and could be isolated with a high yield using one-step affinity chromatography.     

Characterization of β-lactamase enzyme activity in bacterial lysates using MALDI-mass spectrometry

Plasmid-encoded β-lactamases are a major reason for antibiotic resistance in gram negative bacteria. These enzymes hydrolyze the β-lactam ring structure of certain β-lactam antibiotics, consequently leading to their inactivation. The clinical situation demands for specific first-line antibiotic therapy combined with a quick identification of bacterial strains and their antimicrobial susceptibility. Strategies for the identification of β-lactamase activity are often cumbersome and usually lack sensitivity and specificity. The current work demonstrates that matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) is an ideal tool for these analytical investigations. Herein, we describe a fast and specific assay to determine β-lactamase activity in bacterial lysates. The feasibility of the analytical read-out was demonstrated on a MALDI-triple quadrupole (QqQ) and a MALDI time-of-flight (TOF) instrument, and the results allow the comparison of both approaches. The assay specifically measures enzyme-mediated, time-dependent hydrolysis of the β-lactam ring structure of penicillin G and ampicillin and inhibition of hydrolysis by clavulanic acid for clavulanic acid susceptible β-lactamases. The assay is reproducible and builds the basis for future in-depth investigations of β-lactamase activity in various bacterial strains by mass spectrometry.     

The 2.0 A crystal structure of cephalosporin acylase

BACKGROUND: Semisynthetic cephalosporins are primarily synthesized from 7-aminocephalosporanic acid (7-ACA), which is usually obtained by chemical deacylation of cephalosporin C (CPC). The chemical production of 7-ACA includes, however, several expensive steps and requires thorough treatment of chemical wastes. Therefore, an enzymatic conversion of CPC to 7-ACA by cephalosporin acylase is of great interest. The biggest obstacle preventing this in industrial production is that cephalosporin acylase uses glutaryl-7ACA as a primary substrate and has low substrate specificity for CPC. RESULTS: We have solved the first crystal structure of a cephalosporin acylase from Pseudomonas diminuta at 2.0 A resolution. The overall structure looks like a bowl with two "knobs" consisting of helix- and strand-rich regions, respectively. The active site is mostly formed by the distinctive structural motif of the N-terminal (Ntn) hydrolase superfamily. Superposition of the 61 residue active-site pocket onto that of penicillin G acylase shows an rmsd in Calpha positions of 1.38 A. This indicates structural similarity in the active site between these two enzymes, but their overall structures are elsewhere quite different. CONCLUSION: The substrate binding pocket of the P. diminuta cephalosporin acylase provides detailed insight into the ten key residues responsible for the specificity of the cephalosporin C side chain in four classes of cephalosporin acylases, and it thereby forms a basis for the design of an enzyme with an improved conversion rate of CPC to 7-ACA. The structure also provides structural evidence that four of the five different classes of cephalosporin acylases can be grouped into one family of the Ntn hydrolase superfamily.     

A disulfide bridge near the active site of carbapenem-hydrolyzing class A β-lactamases might explain their unusual substrate profile

Bacterial resistance to β-lactam antibiotics, a clinically worrying and recurrent problem, is often due to the production of β-lactamases, enzymes that efficiently hydrolyze the amide bond of the β-lactam nucleus. Imipenem and other carbapenems escape the activity of most active site serine β-lactamases and have therefore become very popular drugs for antibacterial chemotherapy in the hospital environment. Their usefulness is, however, threatened by the appearance of new β-lactamases that efficiently hydrolyze them. This study is focused on the structure and properties of two recently described class A carbapenemases, produced by Serratia marcescens and Enterobacter cloacae strains and leads to a better understanding of the specificity of β-lactamases. In turn, this will contribute to the design of better antibacterial drugs. Three-dimensional models of the two class A carbapenemases were constructed by homology modeling. They suggested the presence, near the active site of the enzymes, of a disulfide bridge (C69-C238) whose existence was experimentally confirmed. Kinetic parameters were measured with the purified Sme-1 carbapenemase, and an attempt was made to explain its specific substrate profile by analyzing the structures of minimized Henri-Michaelis complexes and comparing them to those obtained for the “classical” TEM-1 β-lactamase. The peculiar substrate profile of the carbapenemases appears to be strongly correlated with the presence of the disulfide bridge between C69 and C238. Proteins 27:47–58 © 1997 Wiley-Liss, Inc.     

Beta-lactamases and bacterial resistance to antibiotics

The efficiency of β-lactam antibiotics, which are among our most useful chemotherapeutic weapons, is continuously challenged by the emergence of resistant bacterial strains. This is most often due to the production of β-lactamases by the resistant cells. These enzymes inactivate the antibiotics by hydrolysing the β-lactam amide bond. The elucidation of the structures of some β-lactamases by X-ray crystallography has provided precious insights into their catalytic mechanisms and revealed unsuspected similarities with the DD-transpeptidases, the bacterial enzymes which constitute the lethal targets of β-lactams. Despite numerous kinetic, structural and site-directed mutagenesis studies, we have not completely succeeded in explaining the diversity of the specificity profiles of β-lactamases and their surprising catalytic power. The solutions to these problems represent the cornerstones on which better antibiotics can be designed, hopefully on a rational basis.     

Structural relationship between the active sites of β-lactam-recognizing and amidase signature enzymes: convergent evolution?

The β-lactam-recognizing enzymes (BLRE) make up a superfamily of largely bacterial proteins that include, principally, the dd-peptidases and β-lactamases. The former enzymes catalyze the final step in bacterial cell wall biosynthesis and are inhibited by β-lactam antibiotics, while the latter enzymes catalyze the hydrolytic destruction of β-lactams and represent a major source of bacterial resistance to these antibiotics. The active site of this superfamily of enzymes includes a Ser1/Ser2(Tyr)/Lys1(His)/Lys2 tetrad in which Ser1 is a nucleophilic catalyst that becomes acylated in the formation of an acyl-enzyme intermediate. An oxyanion hole is also present. The amidase signature (AS) enzymes represent another serine amidohydrolase superfamily with no overall structural resemblance to the BLRE. The active site is characterized by a Ser1/Ser2/Lys1/NH tetrad and an oxyanion hole. We point out that there is a close spatial overlap between the two tetrads and speculate that this has arisen from a process of convergent evolution driven by a mechanistic imperative. Conversion of the backbone NH group of the AS tetrad into Lys2 of the BLRE is rationalized and leads to another mechanistic possibility that may dominate BLRE catalysis. The active site triads of other serine amidohydrolases are also briefly and comparatively discussed.     

An amino acid position at crossroads of evolution of protein function: antibiotic sensor domain of BlaR1 protein from Staphylococcus aureus versus clasS D β-lactamases

The integral membrane protein BlaR1 of Staphylococcus aureus senses the presence of β-lactam antibiotics in the milieu and transduces the information to its cytoplasmic side, where its activity unleashes the expression of a set of genes, including that for BlaR1 itself, which manifest the antibiotic-resistant phenotype. The x-ray structure of the sensor domain of this protein exhibits an uncanny similarity to those of the class D β-lactamases. The former is a membrane-bound receptor/sensor for the β-lactam antibiotics, devoid of catalytic competence for substrate turnover, whereas the latter are soluble periplasmic enzymes in gram-negative bacteria with avid ability for β-lactam turnover. The two are clearly related to each other from an evolutionary point of view. However, the high resolution x-ray structures for both by themselves do not reveal why one is a receptor and the other an enzyme. It is documented herein that a single amino acid change at position 439 of the BlaR1 protein is sufficient to endow the receptor/sensor protein with modest turnover ability for cephalosporins as substrates. The x-ray structure for this mutant protein and the dynamics simulations revealed how a hydrolytic water molecule may sequester itself in the antibiotic-binding site to enable hydrolysis of the acylated species. These studies document how the nature of the residue at position 439 is critical for the fate of the protein in imparting unique functions on the same molecular template, to result in one as a receptor and in another as a catalyst.     

Efficient synthesis of β-lactam antibiotics with very low product hydrolysis by a mutant Providencia rettgeri penicillin G acylase

Applied Microbiology and Biotechnology - Penicillin G acylase (PGA) was isolated from Providencia rettgeri PX04 (PrPGApx04) and utilized for the kinetically controlled synthesis of β-lactam...     

Immobilization of the acylase from Escherichia coli on glyoxyl-agarose gives efficient catalyst for the synthesis of cephalosporins

The catalytic properties of penicillin G acylase (PGA) from Escherichia coli, when used in kinetically controlled N-acylation (kcNa) of cephalosporanic nuclei, can be strongly influenced by the moiety in 3-position of the cephem structure. In the synthesis of Cefonicid (1c), the adsorption of the cephalosporanic nucleus (7-SACA) in the PGA active site appeared sensitively increased by a positive ionic interaction between an arginine (ArgA145) in the enzyme active site and the sulphonic group of the β-lactam structure. Interestingly, when PGA was immobilized on solid supports, any effect depending on the substrate structure resulted minimized; the catalytic properties of this enzyme were affected with different outcomes depending on the type of matrix and binding chemistry. The PGA immobilized on glyoxyl-agarose (hydrophilic support activated with aldehyde groups) resulted in a good catalyst when used in kinetically controlled N-acylation of different cephalosporanic nuclei. This derivatives allow much better Vs/Vh(1) (defined as the ratio between the rate of synthesis and the rate of hydrolysis of the acylating agent) than the same enzyme immobilized on Eupergit C, an acrylic hydrophobic supports activated with epoxy groups. The synthetic performances of the Eupergit derivative versus different nuclei were always much poorer if compared with glyoxyl-agarose or the soluble protein. The use of PGA immobilized on glyoxyl-agarose allowed the development of efficient processes for the preparation of Cefazolin in high yield and purity. The results obtained in the optimization of this process are presented.     

Crystal structures of the apo form of β-fructofuranosidase from Bifidobacterium longum and its complex with fructose

We solved the 1.8 ? crystal structure of β-fructofuranosidase from Bifidobacterium longum KN29.1 - a unique enzyme that allows these probiotic bacteria to function in the human digestive system. The sequence of β-fructofuranosidase classifies it as belonging to the glycoside hydrolase family 32 (GH32). GH32 enzymes show a wide range of substrate specificity and different functions in various organisms. All enzymes from this family share a similar fold, containing two domains: an N-terminal five-bladed β-propeller and a C-terminal β-sandwich module. The active site is located in the centre of the β-propeller domain, in the bottom of a 'funnel'. The binding site, -1, responsible for tight fructose binding, is highly conserved among the GH32 enzymes. Bifidobacterium longum KN29.1 β-fructofuranosidase has a 35-residue elongation of the N-terminus containing a five-turn α-helix, which distinguishes it from the other known members of the GH32 family. This new structural element could be one of the functional modifications of the enzyme that allows the bacteria to act in a human digestive system. We also solved the 1.8 ? crystal structure of the β-fructofuranosidase complex with β-D-fructose, a hydrolysis product obtained by soaking apo crystal in raffinose.     

Structural and functional analysis of a conjugated bile salt hydrolase from Bifidobacterium longum reveals an evolutionary relationship with penicillin V acylase

Bile salt hydrolase (BSH) is an enzyme produced by the intestinal microflora that catalyzes the deconjugation of glycine- or taurine-linked bile salts. The crystal structure of BSH reported here from Bifidobacterium longum reveals that it is a member of N-terminal nucleophil hydrolase structural superfamily possessing the characteristic alphabetabetaalpha tetra-lamellar tertiary structure arrangement. Site-directed mutagenesis of the catalytic nucleophil residue, however, shows that it has no role in zymogen processing into its corresponding active form. Substrate specificity was studied using Michaelis-Menten and inhibition kinetics and fluorescence spectroscopy. These data were compared with the specificity profile of BSH from Clostridium perfrigens and pencillin V acylase from Bacillus sphaericus, for both of which the three-dimensional structures are available. Comparative analysis shows a gradation in activity toward common substrates, throwing light on a possible common route toward the evolution of pencillin V acylase and BSH.     

Neutron Diffraction Studies of a Class A β-Lactamase Toho-1 E166A/R274N/R276N Triple Mutant

β-Lactam antibiotics have been used effectively over several decades against many types of bacterial infectious diseases. However, the most common cause of resistance to the β-lactam antibiotics is the production of β-lactamase enzymes that inactivate β-lactams by rapidly hydrolyzing the amide group of the β-lactam ring. Specifically, the class A extended-spectrum β-lactamases (ESBLs) and inhibitor-resistant enzymes arose that were capable of hydrolyzing penicillins and the expanded-spectrum cephalosporins and monobactams in resistant bacteria, which lead to treatment problems in many clinical settings. A more complete understanding of the mechanism of catalysis of these ESBL enzymes will impact current antibiotic drug discovery efforts. Here, we describe the neutron structure of the class A, CTX-M-type ESBL Toho-1 E166A/R274N/R276N triple mutant in its apo form, which is the first reported neutron structure of a β-lactamase enzyme. This neutron structure clearly reveals the active-site protonation states and hydrogen-bonding network of the apo Toho-1 ESBL prior to substrate binding and subsequent acylation. The protonation states of the active-site residues Ser70, Lys73, Ser130, and Lys234 in this neutron structure are consistent with the prediction of a proton transfer pathway from Lys73 to Ser130 that is likely dependent on the conformation of Lys73, which has been hypothesized to be coupled to the protonation state of Glu166 during the acylation reaction. Thus, this neutron structure is in agreement with a proposed mechanism for acylation that identifies Glu166 as the general base for catalysis.     

3-mercapto-1,2,4-triazoles and N-acylated thiosemicarbazides as metallo-β-lactamase inhibitors

The production of β-lactamases is an effective strategy by which pathogenic bacteria can develop resistance against β-lactam antibiotics. While inhibitors of serine-β-lactamases are widely used in combination therapy with β-lactam antibiotics, there are no clinically available inhibitors of metallo-β-lactamases (MBLs), and so there is a need for the development of such inhibitors. This work describes the optimisation of a lead inhibitor previously identified by fragment screening of a compound library. We also report that thiosemicarbazide intermediates in the syntheses of these compounds are also moderately potent inhibitors of the IMP-1 MBL from Pseudomonas aeruginosa. The interactions of these inhibitors with the active site of IMP-1 were examined using in silico methods.     

Biotechnological advances on Penicillin G acylase: Pharmaceutical implications,unique expression mechanism and production strategies

In light of unrestricted use of first-generation penicillins, these antibiotics are now superseded by their semisynthetic counterparts for augmented antibiosis. Traditional penicillin chemistry involves the use of hazardous chemicals and harsh reaction conditions for the production of semisynthetic derivatives and, therefore, is being displaced by the biosynthetic platform using enzymatic transformations. Penicillin G acylase (PGA) is one of the most relevant and widely used biocatalysts for the industrial production of β-lactam semisynthetic antibiotics. Accordingly, considerable genetic and biochemical engineering strategies have been devoted towards PGA applications. This article provides a state-of-the-art review in recent biotechnological advances associated with PGA, particularly in the production technologies with an emphasis on using the Escherichia coli expression platform.