The discovery of penicillin The synthetic sulphonamides were developed in the 1930s. However, bacteria quickly developed resistance to the sulphonamid

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1 19 1/3 20 4% Bstatic, is a biological or chemical agent that stops bacteria from reproducing, while not necessarily harming them otherwise. Adapted from Brett Heintz, PharmD, BCPS The impact of penicillin on therapeutic outcomes The widespread use of penicillin penicillin saved tens of millions of lives around the world since its debut in World War II. The development of antibiotics and their availability for widespread therapeutic use led to a significant new p approach for infection control and health care. Penicillin represents the first defined therapeutic product isolated from microbial sources and is the forerunner for development of all antibiotics to date. of antibiotics to date The technologies developed for the production of penicillin, including microbial strain improvement and chemical engineering methods to mass produce fermentation products, have provided the basis for production of all classes production of all classes of antibiotics to date. The development of to development penicillin represents the combined efforts of several diverse scientific disciplines, including chemistry, biochemistry, microbiology, and chemical engineering. Development and production of many new bioproducts production of many new with diverse clinical applications including applications including cancer, auto immune diseases, new vaccines, etc. are dependent on the same microbial strain selection and large scale chemical engineering methods that were first pioneered for penicillin. The development of penicillin during World War II marked a fundamental change in the p g relationship between government, private industry, and universities for collaboration and funding to support medical research. 第 1 頁

2 The discovery of penicillin The synthetic sulphonamides were developed in the 1930s. However, bacteria quickly developed resistance to the sulphonamides, their spectrum of activity was small, and their side effects were severe. For example, the sulphonamides could not be used to treat venereal disease such as syphilis. Penicillin was accidently discovered in 1928 by Dr. Alexander Fleming in London. At that time, Fleming, a physician, observed that some of his culture dishes containing Staphylococcus aureus( ) were contaminated with a ed so e o s tu e d s es co ta y ococcus au eus( ) e e co ta ated t a mold. This pathogenic bacterium was destroyed by the mold identified as Penicillium notatum. He also noted that filtrates of the mold lysed the staphylococci and were nontoxic in animals. He named the agent Penicillin. Penicillin was a difficult entity to work with because its chemical instability made it difficult to isolate and characterize. Attempts to isolate penicillin were made in the early 1930s by a number of British chemists, but the instability of the substance frustrated their efforts. This formidable challenge was overcome in 1939 when a group at the Sir William Dunn School of Pathology at Oxford University, composed of Howard W. Florey, Ernst B. Chain, Norman y y Heatley and Edward Abraham, and their colleagues, came upon the scene. They were able to prepare a stable form of penicillin and demonstrate its remarkable antibacterial activity along with a lack of toxicity in mice. The Oxford group also determined penicillin's chemical structure in The large scale production of penicillin includes large volume fermentation, large volume extraction, and scaleproductionofpenicillin includes fermentation extraction and purification of the antibiotic which are the foundation for all modern bioreactors and the products produced with this technology. the Nobel Prize in Medicine for 1945 was awarded to Alexander Fleming, Ernst Chain, and Howard Florey. the lactam group of compounds is the most successful example of natural product application and chemotherapy. The lactams and other classes of antibiotics developed over the years have become the key weapons to defeat pestilence. Genetics, fermentation, and biosynthesis of penicillins Fleming's original strain produced only traces of penicillin. Therefore, early brute force genetic manipulations concentrated heavily on the production of mutants and the study of their properties. A strain isolated from a moldy cantaloupe was capable of producing 60 mg/l. df ld l f d NRRL , a spontaneous sector mutant of asingle spore isolations, produced 150 mg/l. X ray treatment increase the yield further to 300 mg/l. t t th i th t A ultraviolet induced mutants, Q 176, produced 550 mg/l, became the ancestor of all of the strains used in industry. Today's s industrial strains produce over 70 g/l. strains over Penicillin had been originally produced in surface culture, but titers were very low. By the use of submerged culture method, strain improvement and medium modifications, such as the use of corn steep liquor as additive, the yield of penicillin was increased by 100 use corn steep additive penicillin increased fold. By the 1950s, it was realized the P. chrysogenum could use additional acyl compounds as side chain chain precursors (other than phenylacetic than phenylacetic acid for penicillin G) and produce new and produce new penicillins, and one of these, penicillin V (phenoxymethylpenicillin), achieved commercial success. 第 2 頁

3 The physiology of P. chrysogenum in relation to penicillin productionto production In commercial production, the usual medium had been a complex one, composed of glucose, corn steep liquor, side chain precursor (phenylacetic acid for penicillin G or phenoxyacetic acid for penicillin V), and mineral salts. glucose to be excellent for growth but poor for penicillin formation, while lactose showed the opposite pattern. (1) devised a medium containing both sugars in which growth occurred at the expense of glucose, and when it was exhausted, the mass of cells began to produce the antibiotic at the expense of lactose. (2) intermittent or continuous feeding of the less expensive glucose could replace batch feeding of the expensive glucose could replace of lactose. biosynthesis of penicillin from its precursors lysine was a potent inhibitor t i of penicillin biosynthesis illi i butthe inhibition could be hibiti b reversed by L aminoadipic acid. the tripeptide ( aminoadipyl) cysteinylvaline (ACV) as an intracellular compound in P. chrysogenum.l aminoadipic chrysogenum. L acid as an important precursor of all penicillinsas an important precursor of all penicillins the tripeptide ( L aminoadipyl) Lcysteinyl D valine (LLD ACV) was established as the crucial intermediate of penicillin biosynthesis. The feedback inhibition by lysine was later proven to be the initial step of lysine biosynthesis in fungi, i.e., the homocitrate synthase reaction. The newer lactams During the 1950s, the future of penicillins G and V became doubtful as resistant strains of S. aureus emerged in hospital populations. The penicillins described up to this point were solvent soluble, exhibiting a high degree of activity against Gram high activity against Gram positive organisms but were much less active against organisms much active against Gram negatives. the compound (6 aminopenicillanic acid, 6 APA) which was the penicillin nucleus was used to make semisynthetic (chemically modified) dt th ti ( h i difi d) versions of penicillin with the beneficial properties of resistance to penicillinase and acid, plus broad spectrum antibacterial activity. the discovery of a completely different type of penicillin, a hydrophilic type, showing equivalent activity against both classes of microorganisms (i.e., penicillin N) In 1948, Giuseppe Brotzu isolated Cephalosporium h l acremonium, a cephalosporin producing fungus, from sewage. Brotzu's strain produced two antibiotics, i.e., cephalosporin P, active only against Gram positive organisms and cephalosporin N, which was active against both Grampositive a c as act e aga st Ga post eand Gram dg a negative bacteria. Cephalosporin P was found to be of steroidal nature and not a lactam at all. Cephalosporin N, on the other hand, was found to be a true penicillin possessing an D aminoadipyl side chain. It was renamed penicillin N. In comparison to penicillin G, it was only 1% as active against illi i i illi it l ti i t Gram positive forms but had equal to or somewhat greater activity against Gramnegative bacteria. However, it never became a commercial antibiotic. The hydrophilic nature of penicillin N and its roughly equivalent activity against p y y g Gram positive and Gram negative bacteria was due to the carboxyl group in the side chain. 第 3 頁

4 More lactams A second antibiotic produced by Brotzu's strain of A. chrysogenum was found to be related to penicillin N in that it consisted of a lactam ring attached to a side chain which was identical to that of penicillin N, i.e., D a aminoadipic acid. It differed, however, from the penicillins in containing a six membered dihydrothiazine ring in place of the five membered thiazolidine ring of the penicillins. It was called cephalosporin C. Thus, the era of the cephalosporins was launched. Cephalosporin C strongly absorbed ultraviolet light, was stable to acid and to penicillin lactamase, was non toxic, C l b lt i l t li ht t bl t id d t i illi l t t i and had in vivo activity in mice. It had only 0.1% of the activity of penicillin G against sensitive staphylococci, although its activity against Gram negative bacteria equaled that of penicillin G. Although neither penicillin N nor cephalosporin C was ever commercialized, they led to important knowledge on the biosynthesis of these compounds and the development of many powerful semisynthetic cephalosporins of great use in medicine. The nucleus of cephalosporin C was named 7 aminocephalosporanic acid (7 ACA). By chemical removal of its D aaminoadipic acid side chain and replacement with phenylacetic chain and phenylacetic acid, a penicillinase resistant semi synthetic a semi synthetic compound was obtained which was 100 times as active as cephalosporin C. Many other new cephalosporins with wide antibacterial spectra were developed in the ensuing years, e.g., cephalothin, cephaloridine, and cephaloglycin, making the semisynthetic cephalosporins the most important group of antibiotics at that time. The stability of the cephalosporins to penicillinase was evidently a function of its dihydrothiazine ring since (1) the D aminoadipic acid side chain did not render penicillin N immune to attack and (2) removal of the acetoxy chain did not immune to attack and removal of the acetoxy group from cephalosporin C did not decrease its stability to penicillinase. Cephalosporin C competitively inhibited the action of penicillinase from Bacillus cereus on penicillin G. Although it did not have a similar effect on the S. aureus enzyme, certain of its derivatives did. Another advantage was that cephalosporins could be given to some patients who were allergic to penicillins More lactams: to ravel how penicillin N and cephalosporin C were made From a biosynthetic sense, the relationship of penicillin N and cephalosporin C was of great interest. Use protoplast lysates of A. chrysogenum to convert labeled valine into a penicillin. discoveryat discovery at M.I.T. in 1976 of the ring expansion reaction (Kohsaka M.I.T.in1976 ofthe ring expansion reaction and Demain 1976), catalyzed by the expandase (deacetoxycephalosporin C synthase, DAOC synthase) enzyme. cephalosporins were produced from a penicillin. (For many years, it had been thought that penicillin N and cephalosporin C were products of different biosynthetic branches in A. chrysogenum. ) penicillin N + 2 oxoglutarate + O 2 deacetoxycephalosporin C + succinate + CO 2 + H 2 O cell free activity of the enzyme from A. chrysogenum formed the important tripeptide precursor of all penicillins and cephalosporins, i.e., (L aminoadipyl) L cysteinyl D valine hl i i di l) L ti ld li (LLD ACV). The enzyme ACV synthetase t was proven to be a single multifunctional enzyme acting on L aminoadipic acid, L cysteine, and L valine to produce LLDACV. 3ATP+ 3 ATP L 2 aminohexanedioate aminohexanedioate + L cysteine cysteine + L valine + H2O 3 AMP 3AMP+ 3 PPi3 + N N [L 5 amino 5 carboxypentanoyl] L 5 L cysteinyl D valine (PPi: diphosphate) isolation of pure isopenicillin N synthetase (IPN synthase, cyclase ) which converted the LLD ACV to isopenicillin N. The long held notion that lactams were produced only by fungi was shattered by a report from Merck & Co. that a streptomycetea producedpenicillin produced penicillin N. The discovery of cephamycin C led to much research on and development of prokaryotic cephalosporins since the presence of the methoxy group on the on lactam ring made the molecule more active made the molecule more active against Gram negative and anaerobic pathogens and more resistant to Gram negative lactamases. 第 4 頁

5 Biosynthetic pathway to penicillin, cephalosporin, and cephamycinsand cephamycins Overall, there are three main and important steps to the biosynthesis of penicillin G (benzylpenicillin). The first step is the condensation of three amino acids L aminoadipic acid, L cysteine, L valine into a tripeptide. Before condensing into the tripeptide, the amino acid L valine must undergo epimerization to become D valine. The condensed tripeptide is named (L aminoadipyl) L cysteine D valine (ACV). The condensation reaction and epimerization are both catalyzed by the enzyme (L aminoadipyl) L cysteine D valine synthetase (ACVS), a nonribosomal peptide synthetase or NRPS. The second step in the biosynthesis of penicillin G is the oxidative conversion of linear ACV into the bicyclic intermediate isopenicillin N by isopenicillin N synthase (IPNS), which is encoded by the gene pcbc. Isopenicillin is Isopenicillin N is a very weak intermediate, because it does not show strong antibiotic activity. The final step is a transamidation by isopenicillin N N acyltransferase, in which the the aminoadipyl side chain of isopenicillin N is removed and exchanged for a phenylacetyl side chain. This reaction is encoded by the gene pende, which is unique in the process of obtaining penicillins Like fungal cephalosporin C, cephamycin C was never used clinically but was employed for semi synthesis of many medically useful compounds. A more potent semi synthetic cephamycin, cefoxitin, was rapidly commercialized by Merck, to be followed later by cefmetazole, temocillin, cefotetan, and other semi synthetic cephalosporins. 123 More lactams: lactams: discovered inhibitors of inhibitors of lactamase In the 1970s and 1980s, the pathways to the penicillins and the cephalosporins including cephamycin C were worked out. Late in the 1970s came reports on the production of lactam antibiotics which were neither penicillins nor cephalosporins. The most important was clavulanic acid from streptomycetes, which possessed only weak antibiotic activity but was an excellent inhibitor possessed only weak antibiotic activity but was an excellent inhibitor of lactamase. It became a blockbuster product by being coformulated with broad spectrum semi synthetic penicillins which were susceptible to lactamase, e.g., with amoxicillin; the combination was known as Augmentin (trade name). the discovery at Merck of the carbapenems The first, called thienamycin, was discoveredby a screening protocol based on inhibition of peptidoglycan synthesis. The on of peptidoglycan synthesis. antibiotic was produced by Streptomyces cattleya, which also made cephamycin C. Carbapenems resembled the penicillins inhaving a lactam ring fused to a five membered ring. They differed in that the dt fi d i diff di th tth five membered ring was unsaturated and contained a carbon atom instead of sulfur. Sulfur was, however, present in another location in all the carbapenems produced by streptomycetes. p p y thienamycin is the most potent, most broad spectrum, and non toxic natural antibacterial agent ever found. It inhibited cell wall synthesis, as did the penicillins and cephalosporins, and was relatively resistant to microbial lactamases. resistant to lactamases 第 5 頁

6 Mode of action against microbials Penicillin binding protein (PBP) are responsible responsible for cross linking in the bacterial cell wall. They make peptide bonds between lysine and alanine. Penicillin, Cephalosporins, and carbapenems bind into the reaction site of PBP s rendering the enzyme unable to cross link the bacterial wall giving bactericidal activity. The productions of lactams Penicillin is a secondary metabolite of certain species of Penicillium and is produced when growth of the fungus is inhibited by stress. It is not produced during active growth. Production is also limited by feedback in the synthesis pathway of penicillin. penicillin ketoglutarate + AcCoA homocitrate L aminoadipic acid L lysine + lactam The by product, l lysine, inhibits the production of homocitrate, so the presence of exogenous lysine should be l hb h d fh h f l h ldb avoided in penicillin production. The Penicillium cells are grown using a technique called fed batch culture, in which the cells are constantly g subject to stress, which is required for induction of penicillin production. The available carbon sources are also important: Glucose inhibits penicillin production, whereas lactose does not. The ph and the levels of nitrogen, lysine, phosphate, and oxygen of the batches must also be carefully controlled. The biotechnological method of directed evolution has been applied to produce by mutation a large number of Penicillium strains. These techniques include error prone PCR, DNA shuffling, ITCHY, and strand overlap PCR. Semisynthetic penicillins are prepared starting from the penicillin nucleus 6 APA and 7 ACA. 第 6 頁

7 PENICILLIN: structure and mothod of action The penicillins are natural or synthetic antibacterial agents derived from fungi. All penicillins share three basic chemical components: a thiazoldine ring, an attached beta attached beta lactam ring, and a side chain. and a chain Other antibiotics that share the characteristic beta lactam structure with penicillin include the cepahalosporins, carbapenamines, and monobactams. Collectively, these antibiotic classes are often referred these antibiotic classes are often referred to as beta lactams. (beta lactam backbone) All penicillins are bacteriocidal to actively growing and dividing organisms. They inhibit cell wall synthesis by binding to the enzymes that produce the protein crosslinks, which form the bacterial cell wall. These enzymes, located just beneath the cell walls, are also referred to as penicillin binding proteins. The resulting structural weakness is compounded by activation of autolytic enzymes, causing lysisof the susceptible bacteria. The ability of the cell wall to be penetrated i tibl b t i bilit f ll ll t b t t and the attraction of the cellular enzymes to penicillin which when bound to them, inhibits their function are major factors in the susceptibility of bacteria to penicillin. Bacterial resistance to penicillin has been a problem since its introduction and has increased in resistance to a problem since its introduction and increased in recent years. Resistance may be natural or acquired. Certain organisms, particularly members of the staphylococcus sp. have the ability to secrete beta lactamase, an enzyme that destroys the beta lactam nucleus and thereby inactivates the penicillin molecule and thereby the molecule Acquired resistance to penicillin occurs through several mechanisms including spontaneous mutation of bacterial chromosome(s), recombination of DNA, or exchange of genetic materials with other bacteria. Given all mechanisms, the primary acquired resistance pattern to the acquired resistance pattern to penicillins of most import clinically is the ability to produce beta lactamase. Spectrum of activity The natural penicillins are primarily effective against baerobic, gram positive organisms such as streptococi, enterococci, and some staphylococci that do not produce beta lactamase. Newer synthetic penicillins such as the aminopenicillins aminopenicillins and extended spectrum penicillins have increased this spectrum to include activity against some gram negative organisms such as H influenza, N gonorrhoeae, and E coli that have not developed resistance. The addition of beta lactamase inhibitors to some aminopenicillins further increases the spectrum of activity making the penicillin family one of the spectrum activity making penicillin family one broadest spectrum class of antibiotics. Despite increased bacterial resistance and the development of newer antibiotic classes, penicillin remains one of the most useful antibiotics available today. 第 7 頁

8 Natural Penicillin (1) penicillin G (benzylpenicillin) for parenteral use; have the narrowest spectrum of activity. p easily inactivated by gastric secretions. ineffective against beta lactamase producing organisms. available in four salt forms: the potassium and sodium salt forms are interchangable formulations designed to stabilize the penicillin molecule during storage. The benzathine and procaine salt forms are designed for slower absorption and therefore longer duration of action. (2) penicillin V (phenoxymethylpenicillin) stable in gastric secretions. Oral administration of natural penicillin is desirable. achieves relatively low peak plasma levels and shouldbe used only in mild, localized infections caused by susceptible organisms. Penicillin V is still considered the drug of choice for streptococcal pharyngitis Synthetic Penicillin Penicillinase resistance prnicillins This group of drugs achieves their effectiveness by the addition of a large side chain to the penicillin molecule, which prevents penicillinase produced by staphylococcus from entering the penicillin molecule and cleaving the beta lactam ring. Nafcillin oxacillin cloxacillin dicloxacillin The spectrum of activity of these drugs makes them useful in the treatment of mild infections of the skin and soft tissue, especially when penicillinase producing staphylococci are the presumed or known causative agents. agents. Methicillin is the prototype for these drugs, but is no longer produced in this country because the development of methicillin resistant staphylococcus aureus (MRSA) rendered it so ineffective that its relatively severe adverse effects are no longer justified. 第 8 頁

9 Aminopenicillins: broad spectrumn penicillins ampicillin amoxicillin The aminopenicillins were the first group of penicillins to have activity against gram negative bacteria. Adding an amino group to the benzylpenicillin molecule produced ampicillin, the prototype for these drugs. prototype these drugs All three drugs have virtually the same broad spectrum of activity against gram-positive and gram-negative organisms. bacampicillin Aminopenicillin/beta lactamase inhibitor combinations Despite the broader activity spectrum of the aminopenicillins, by themselves, they are ineffective against beta lactamase producing organisms. The addition of beta lactamase inhibitors to the aminopenicillins was a critical step in improving their spectrums of activity. The beta lactamase inhibitors have no intrinsic antimicrobial activity. Rather, these compounds, clavulanic acid, sulbactam, and tazobactam, perform in either of two ways: (1) binding to the active site of the beta lactamase enzyme, thereby preventing its attack on the beta lactam ring, or (2) enhancing the affinity of penicillin binding proteins in the bacteria for the penicillin molecule thereby facilitating breakdown of the bacterial cell wall. clavulanic acid sulbactam tazobactam 第 9 頁

10 Extended spectrum penicillins Further manipulation of the ampicillin molecule has produced additional categories of penicillins with even broader spectrums of activity. The carboxypenicillin group includes carbenicillin and ticarcillin. Ureidopenicillins include mezlocillin and azlocillin. Piperacillin is classified as a piperazine penicillin. The primary indication for the carboxypenicillins is for the treatment of Pseudomonas aeruginosa infections. i Piperacillinand the ureidopenicillins illi id i illi have the widest spectrums of antibacterial activity, h ib i i i and have enhanced anti Pseudomonas activity. carbenicillin ticarcillin mezlocillin azlocillin Extended spectrum penicillins, while useful in the treatment penicillins useful in the treatment of nosocomial( )and other complicated infections, have a higher incidence of adverse effects and should be reserved for complicated situations that are outside the realm of that are outside the realm of primary care. Piperacillin Adverse effects of peicillins The penicillins are generally considered among the safest of antibiotics. With the exception of the newer extendedspectrum drugs, they are classified as FDA pregnancy category B drugs. The incidence of adverse responses to penicillin ranges from percent and may manifest in the id d i illi f d if i immune, nervous, renal, gastrointestinal, integumentary, and hematologic systems. Adverse effects are further subdivided into adverse side effects and allergic reactions. f th id d i t d id t d i ti skin rash is hi the most common adverse effect and allergic and hypersensitivity responses, including anaphylaxis( ), exfoliative dermatitis and Stevens Johnson syndrome are the most serious dermatologic manifestations. Gastrointestinal adverse effects are most common in response to manifestations adverse effects are most commoninresponse ampicillin and include nausea, vomiting, and diarrhea. 第 10 頁

11 Penicillin binding proteins Penicillin binding proteins (PBPs) are a group of proteins that are characterized by their affinity for and binding of penicillin. They are a normal constituent of many bacteria; the name just reflects the way by i fl h b which the protein was discovered. All lactam antibiotics (except for tabtoxinine lactam, which inhibits glutamine synthetase) bind to PBPs, which are essential for bacterial cell wall biogenesis. h t i l ll ll bi i Bacteria often develop resistance to lactam antibiotics by synthesizing a lactamase, an enzyme that attacks the lactam ring. To overcome this resistance, lactamovercome resistance antibiotics are often given with are often lactamase inhibitors such as clavulanic acid. Diversity of PBPs There are a large number of PBPs, usually several in each organism, and they are found as both membrane bound and cytoplasmic proteins. The different PBPs occur in different numbers per cell and have varied affinities for penicillin. The PBPs are usually broadly classified into high molecular y weight (HMW) and low molecular weight (LMW) categories. Function of and antibiotics against PBPs PBPs are all involved in the final stages of the synthesis of peptidoglycan, which is the major component of bacterial cell walls. Bacterial cell wall synthesis is essential to growth, cell division (thus reproduction) and essential to reproduction) and maintaining the cellular structure in bacteria. Inhibition of PBPs leads to irregularities in cell wall structure such as elongation, lesions, loss of selective permeability, and eventual cell death and lysis. and eventual death and lysis PBPs have been shown to catalyze a number of reactions involved in the process of synthesizing cross linked peptidoglycan from lipid intermediates and mediating the removal of D alanine from the precursor of peptidoglycan. pp gy N acetylglucosamine (GlcNAc or NAG) and N acetylmuramic acid (MurNAc or NAM) PBPs bind lactam antibiotics because they are similar in chemical structure to the modular pieces that form the peptidoglycan. When they bind to penicillin, the lactam amide bond is ruptured to form a covalent bond with the catalytic serine residue at the PBPs active site. This is an irreversible reaction and inactivates the enzyme. There has been a great deal of research into PBPs because of their role in antibiotics and resistance. Bacterial cell wall synthesis and the role of PBPs in its synthesis is a very good target for drugs of selective toxicity because the metabolic pathways and enzymes are unique to bacteria. Resistance to antibiotics has come about through overproduction of PBPs and formation of PBPs that have low affinity for penicillins (among other mechanisms such as lactamase production). Research on PBPs has led to the discovery of new semi synthetic lactams, wherein altering the side chains on the original penicillin molecule has increased the affinity of PBPs for penicillin, and, thus, increased effectiveness in illi l l i it f f i illi d i d ti i bacteria with developing resistance. 第 11 頁

12 Penicillin Binding Protein 3 of Pseudomonas Aeruginosa (PDB 3OC2) N terminal domain head sub domain Transpeptidase domain The enzyme has a penicillin insensitive transglycosylase N terminal domain (involved in formation of linear glycan strands) and a penicillin sensitive transpeptidase C terminal domain (involved in cross linking of the peptide subunits) and the serine at the active site is conserved in all members of the PBP family. it d th i t th it i d i b th f il Penicillin binding mode of PBP The enzyme, D alanyl D alanine carboxypeptidase/transpeptidase creates a crosslink between two chains in the peptidoglycan net. The structure contains a molecule specially designed by researchers to capture a snapshot of the process of crosslinking. Cephalosporin, a drug similar to penicillin, is bound directly to the serine in the active site. The drug is in the position normally occupied by one of the strands to be crosslinked, as indicated by the transparent dots that extend to the left. A little peptide has been attached to this drug in the position normally occupied by the other strand in the crosslink. This structure shows how the complex might look just after the crosslink is made. PDB code: 1hvb 第 12 頁

13 Penicillin resistance D-alanyl-D-alanine peptidase from Streptomyces sp. R61 (PDB: 3pte) class A beta-lactamase (penicillinase) of Bacillus licheniformis 749/C (PDB: 4blm) Bacteria have developed many ways to thwart the action of penicillin. Some change the penicillin binding proteins in subtle ways, so that they still perform their function but do not bind to the drugs. Some develop more effective ways to shield the sensitivethey their function not bind to the drugs develop more effective to shield the sensitive enzymes from the drug or methods to pump drugs quickly away from the cell. But the most common method is to create a special enzyme, a beta lactamase (also called penicillinase) that seeks out the drug and destroys it. Many beta lactamases use the same machinery as used by the penicillin binding proteins so similar, in fact, than many researchers y gp, y believe that the beta lactamases were actually developed by evolutionary modification of penicillin binding proteins. The penicillin binding proteins, like the one shown on the left (PDB entry 3pte), use a serine amino acid in their reaction, colored purple here. The serine forms a covalent bond with a peptidoglycan chain, then releases it as it forms the crosslink with another part of the peptidoglycan network. Penicillin binds to this serine but does not release it, thus permanently blocking the active site. Beta lactamases, like the one shown on the right (PDB entry 4blm), have a similar serine in their active site pocket. Penicillin also binds to this serine, but is then released in an inactivated form. Other beta lactamases do the same thing, but use a zinc ion instead of a serine amino acid to inactivate the penicillin. Additional figures Benzathine is a diamine which stabilises penicillin and prolongs its sojourn( ) prolongs its sojourn( ) when injected into tissues. Procaine is a local anesthetic drug of the amino ester group. It was used primarily to reduce the used primarily to reduce the pain of intramuscular injection of penicillin, and it was also used in dentistry. beta lactam plasmid sharing between bacteria Peptidoglycan unit penicillin core illi 第 13 頁

14 Additional figures Energy minimized computational model of peptidoglycan strands bound to Streptomyces R61 PBP 第 14 頁