C-terminal bombesin sequence requirements for binding and effects on protein synthesis in Swiss 3T3 cells.

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class.     

Primary structure of the Streptomyces R61 extracellular DD-peptidase. 1. Cloning into Streptomyces lividans and nucleotide sequence of the gene

An 11,450-base DNA fragment containing the gene for the extracellular active-site serine DD-peptidase of Streptomyces R61 was cloned in Streptomyces lividans using the high-copy-number plasmid pIJ702 as vector. Amplified expression of the excreted enzyme was observed. Producing clones were identified with the help of a specific antiserum directed against the pure DD-peptidase. The coding sequence of the gene was then located by hybridization with a specific nucleotide probe and sub-fragments were obtained from which the nucleotide sequence of the structural gene and the putative promoter and terminator regions were determined. The sequence suggests that the gene codes for a 406-amino-acid protein precursor. When compared with the excreted, mature DD-peptidase, this precursor possesses a cleavable 31-amino-acid N-terminal extension which has the characteristics of a signal peptide, and a cleavable 26-amino-acid C-terminal extension. On the basis of the data of Joris et al. (following paper in this journal), the open reading frame coding for the synthesis of the DD-peptidase was established. Comparison of the primary structure of the Streptomyces R61 DD-peptidase with those of several active-site serine beta-lactamases and penicillin-binding proteins of Escherichia coli shows homology in those sequences that comprise the active-site serine residue. When the comparison is broadened to the complete amino acid sequences, significant homology is observed only for the pair Streptomyces R61 DD-peptidase/Escherichia coli ampC beta-lactamase (class C). Since the Streptomyces R61 DD-peptidase and beta-lactamases of class A have very similar three-dimensional structures [Kelly et al. (1986) Science (Wash. DC) 231, 1429-1431; Samraoui et al. (1986) Nature (Lond.) 320, 378-380], it is concluded that these tertiary features are probably also shared by the beta-lactamases of class C, i.e. that the Streptomyces R61 DD-peptidase and the beta-lactamases of classes A and C are related in an evolutionary sense.     

Site-directed mutagenesis of the Streptomyces R61 DD-peptidase. Catalytic function of the conserved residues around the active site and a comparison with class-A and class-C beta-lactamases.

The importance of various residues in the Streptomyces R61 penicillin-sensitive DD-peptidase has been assessed by site-directed mutagenesis. The replacement of the active Ser62 by a Cys residue yielded an inactive protein which was also unable to recognize penicillin. The activity of the Lys65----Arg mutant with the peptide and thiolester substrates was decreased 100-200-fold and the rate of penicillin inactivation was decreased 20,000-fold or more. The mutant thus behaved as a poor, but penicillin-resistant, DD-peptidase. The other studied mutations, the mutations Phe58----Leu, Tyr90----Asn, Thr101----Asn, Phe164----Ala, Asp225----Glu and Asp225----Asn had little influence on the catalytic and penicillin-binding properties. The Asp225 mutants did not exhibit an increased sensitivity to cefotaxime. The Phe164----Ala mutant was significantly more unstable than the wild-type enzyme.     

Streptomyces K15 DD-peptidase-catalysed reactions with suicide beta-lactam carbonyl donors.

The values of the kinetic parameters that govern the interactions between the Streptomyces K15 DD-peptidase and beta-lactam compounds were determined by measuring the inactivating effect that these compounds exert on the transpeptidase activity of the enzyme and, in the case of [14C]benzylpenicillin and [14C]cefoxitin, by measuring the amounts of acyl-enzyme formed during the reaction. K15 DD-peptidase binds benzylpenicillin or cefoxitin at a molar ratio of 1:1. Benzylpenicilloate is the major product released during breakdown of the acyl-enzyme formed with benzylpenicillin. Benzylpenicillin is not a better acylating agent than the amide Ac2-L-Lys-D-Ala-D-Ala and ester Ac2-L-Lys-D-Ala-D-lactatecarbonyl-donor substrates. beta-Lactam compounds possessing a methoxy group on the alpha-face of the molecule show high inactivating potency.     

Amyloid fibril formation from crude protein mixtures

Amyloid fibrils have potential as bionanomaterials. A bottleneck in their commercial use is the cost of the highly purified protein typically needed as a starting material. Thus, an understanding of the role of heterogeneity in the mixtures from which amyloid fibrils are formed may inform production of these structures from readily available impure starting materials. Insulin, a very well understood amyloid-forming protein, was modified by various reagents to explore whether amyloid fibrils could still form from a heterogeneous mixture of insulin derivatives. Aggregates were characterized by thioflavin T fluorescence and transmission electron microscopy. Using acetylation, reduction carboxymethylation, reduction pyridylethylation, trypsin digestion and chymotrypsin digestion, it was shown that amyloid fibrils can form from heterogeneous mixtures of modified insulin. The modifications changed both the rate of reaction and the yield of the final product, but led to fibrillar structures, some with interesting morphologies. Well defined, long, unbranched fibrils were observed in the crude reduced carboxymethylated insulin mixture and the crude reduced pyridylethylated insulin revealed the formation of "wavy" fibrils, compared with the straighter native insulin amyloid fibrils. Although trypsin digestion inhibited fibrils formation, chymotrypsin digestion of insulin produced a mixture of long and short fibrils under the same conditions. We conclude that amyloid fibrils may be successfully formed from heterogeneous mixtures and, further, that chemical modification may provide a simple means of manipulating protein fibril assembly for use in bionanotechnological applications, enabling some design of overall morphology in the bottom-up assembly of higher order protein structures from amyloid fibrils.     

PROTEOLYTIC DIGESTION OF BOVINE BRAIN WHITE MATTER PROTEOLIPID

—Proteolipids were previously considered to be resistant to proteolytic digestion. In the present study, crude bovine white matter proteolipid, proteolipid apoprotein, and chemically modified proteolipids were subjected to the action of the following proteolytic enzymes in the absence and presence of detergents: trypsin, α-chymotrypsin, elastase, thermolysin and collagenase. The course of digestion was followed by the release of Fluorescamine-reactive groups. Tryptic digestion of the crude proteolipid and the apoprotein in the absence of detergent amounted to 10 and 40% respectively of the digestion in the presence of detergent. Peptide mapping and protein analyses of both the soluble digests and the insoluble residues confirmed digestion. In the presence of either sodium deoxycholate or Triton X-100, essentially all of the crude proteolipid and 60% of the proteolipid apoprotein were solubilized by trypsin. Digestion of the apoprotein was observed only in preparations which had not been dried. In the absence of detergent, (1) the oxidized crude proteolipid was more susceptible to tryptic digestion than were either the unoxidized or carboxymethylated preparations, (2) both the apoprotein and the oxidized proteolipid were digested by thermolysin or α-chymotrypsin, and (3) all preparations were attacked by elastase.     

Studies on the high-sulphur proteins of reduced mohair. The isolation and amino acid sequence of protein scmkb-m1.2.

The complete amino acid sequence of mohair protein, SCMKB-M1.2 (97 residues), was determined. The protein was isolated from reduced and carboxymethylated mohair by chromatography on DEAE-cellulose phosphate. Peptides for sequence determination were obtained by digestion with trypsin, pepsin, chymotrypsin, thermolysin and papain, and were fractionated by DEAE-cellulose chromatography, paper chromatography and electrophoresis. The sequence of the peptides were determined by the Edman degradation method (by use of both the Beckman Sequence and a non-automatic procedure), and by partial acid hydrolysis. The protein is closely homologous to wool protein SCMKB-IIIB2, and also contains acetylated alanine as N-terminal amino acid.     

Toward better antibiotics: crystallographic studies of a novel class of DD-peptidase/beta-lactamase inhibitors

Beta-lactam antibiotics are vital weapons in the treatment of bacterial infections, but their future is under increasing threat from beta-lactamases. These bacterial enzymes hydrolyze and inactivate beta-lactam antibiotics, rendering the host cell resistant to the bactericidal effects of the drugs. Nevertheless, the bacterial D-alanyl-D-alanine transpeptidases (DD-peptidases), the killing targets of beta-lactams, remain attractive targets for antibiotic compounds. Cyclic acyl phosph(on)ates have been developed and investigated as potential inhibitors of both transpeptidases and beta-lactamases. The X-ray crystal structures of the complexes of the Streptomyces strain R61 DD-peptidase inhibited by a bicyclic [1-hydroxy-4,5-benzo-2,6-dioxaphosphorinanone(3)-1-oxide] and a monocyclic [1-hydroxy-4-phenyl-2,6-dioxaphosphorinanone(3)-1-oxide] acyl phosphate were determined to investigate the mode of action of these novel inhibitors. The structures show, first, that these inhibitors form covalent bonds with the active site serine residue of the enzyme and that the refractory complexes thus formed are phosphoryl-enzyme species rather than acyl enzymes. The complexes are long-lived largely because, after ring opening, the ligands adopt conformations that cannot directly recyclize, the latter a phenomenon previously observed with cyclic acyl phosph(on)ates. While the two inhibitors bind in nearly identical conformations, the phosphoryl-enzyme complex formed from the monocyclic compound is significantly less mobile than that formed from the bicyclic compound. Despite this difference, the complex with the bicyclic compound breaks down to regenerate free enzyme somewhat more slowly than that of the monocyclic. This may be because of steric problems associated with the reorientation of the larger bicyclic ligand required for reactivation. The structures are strikingly different in the orientation of the phosphoryl moiety from those generated using more specific phosph(on)ates. Models of the noncovalent complexes of the monocyclic compound with the R61 DD-peptidase and a structurally very similar class C beta-lactamase suggest reasons why the former enzyme is phosphorylated by this compound, while the latter is acylated. Finally, this paper provides information that will help in the design of additional DD-peptidase inhibitors with the potential to serve as leads in the development of novel antibiotics.     

The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family.

Homology searches and amino acid alignments, using the Streptomyces R61 DD-peptidase/penicillin-binding protein as reference, have been applied to the beta-lactamases of classes A and C, the Oxa-2 beta-lactamase (considered as the first known member of an additional class D), the low-Mr DD-peptidases/penicillin-binding proteins (protein no. 5 of Escherichia coli and Bacillus subtilis) and penicillin-binding domains of the high-Mr penicillin-binding proteins (PBP1A, PBP1B, PBP2 and PBP3 of E. coli). Though the evolutionary distance may vary considerably, all these penicillin-interactive proteins and domains appear to be members of a single superfamily of active-site-serine enzymes distinct from the classical trypsin or subtilisin families. The amino acid alignments reveal several conserved boxes that consist of strict identities or homologous amino acids. The significance of these boxes is highlighted by the known results of X-ray crystallography, chemical derivatization and site-directed-mutagenesis experiments.     

The primary structure of the alpha subunit of protocatechuate 3,4-dioxygenase. I. Isolation and sequence of the tryptic peptides.

The carboxymethylated alpha subunit of protocatechuate 3,4-dioxygenase was digested with trypsin. The 14 tryptic peptides were isolated by ion exchange chromatography on DEAE-Sephadex and by gel filtration chromatography. Automated Edman degradation and carboxypeptidase Y and B digestion were used to establish the sequence of these peptides. Further fragmentation of two tryptic peptides, T3 and T5, by Staphylococcus aureus protease and cyanogen bromide, respectively, was necessary to complete the sequences. The tryptic peptides accounted for a minimum of 199 residues out of a total of 202 residues predicted by amino acid analysis.     

The D-methyl group in beta-lactamase evolution: evidence from the Y221G and GC1 mutants of the class C beta-lactamase of Enterobacter cloacae P99

The beta-lactam antibiotics act through their inhibition of D-alanyl-D-alanine transpeptidases (DD-peptidases) that catalyze the last step of bacterial cell wall synthesis. Bacteria resist beta-lactams by a number of mechanisms, one of the more important of which is the production of beta-lactamases, enzymes that catalyze the hydrolysis of these antibiotics. The serine beta-lactamases are evolutionary descendants of DD-peptidases and retain much of their structure, particularly at the active site. Functionally, beta-lactamases differ from DD-peptidases in being able to catalyze hydrolysis of acyl-enzyme intermediates derived from beta-lactams and being unable to efficiently catalyze acyl transfer reactions of D-alanyl-D-alanine terminating peptides. The class C beta-lactamase of Enterobacter cloacae P99 is closely similar in structure to the DD-peptidase of Streptomyces R61. Previous studies have demonstrated that the evolution of the beta-lactamase, presumably from an ancestral DD-peptidase similar to the R61 enzyme, included structural changes leading to rejection of the D-methyl substituent of the penultimate D-alanine residue of the DD-peptidase substrate. This seems to have been achieved by suitable placement of the side chain of Tyr 221 in the beta-lactamase. We show in this paper that mutation of this residue to Gly 221 produces an enzyme that more readily hydrolyzes and aminolyzes acyclic D-alanyl substrates than glycyl analogues, in contrast to the wild-type beta-lactamase; the mutant is therefore a more efficient DD-peptidase. Molecular modeling showed that the D-alanyl methyl group fits snugly into the space originally occupied by the Tyr 221 side chain and, in doing so, allows the bound substrate to assume a conformation similar to that on the R61 DD-peptidase, which has a hydrophobic pocket for this substituent. Another mutant of the P99 beta-lactamase, the extended spectrum GC1 enzyme, also has space available for a D-alanyl methyl group because of an extended omega loop. In this case, however, no enhancement of activity against D-alanyl substrates with respect to glycyl was observed. Accommodation of the penultimate D-alanyl methyl group is therefore necessary for efficient DD-peptidase activity, but not sufficient.     

Hydrolysis of polypeptides and proteins utilizing a mixture of dipeptidylaminopeptidases with analysis by GC/MS.

The hydrolysis of polypeptides and proteins to dipeptides using a mixture of dipeptidylaminopeptidase I and IV is demonstrated for several compounds. The dipeptide products were identified by gas chromatography/mass spectrometry of their acyl, ester derivatives. Partial hydrolysis of carboxymethylated soybean trypsin inhibitor (mw 20,327) shows the viability of the methods with an intact protein. Polypeptides with N-terminal arginyl or lysyl residues, which cannot be hydrolyzed by dipeptidylaminopeptidase I alone, are readily hydrolyzed by the dipeptidylaminopeptidase $\text{I}\text{IV}$ mixture, as shown by the digestion of ribonuclease S-peptide. The enzyme mixture releases dipeptides from the N-terminus of a polypeptide or protein with hydrolysis being terminated only when proline appears in position three of a remaining polypeptide fragment. The techniques are discussed in terms of new types of analyses which can be performed using this combined enzyme-GC/MS method.     

Purification and primary structure of proteinous alpha-amylase inhibitor from Streptomyces chartreusis.

A new polypeptide inhibitor, AI-409, that inhibits human salivary alpha-amylase, was purified from a fermentation broth of Streptomyces chartreusis strain No. 409. This protein consists of a single-chain polypeptide of 78 amino acid residues, and includes two disulfide bridges. The primary structure of AI-409 and the locations of the disulfide bridges were identified by enzymatic digestion and the automatic Edman technique. Enzymatic digestion was done with trypsin, carboxypeptidase Y, and chymotrypsin. One of the disulfide bridges was between Cys(10) and Cys(26), and the other between Cys(44) and Cys(71).     

The relationship to knobs of the 92,000 D protein specific for knobby strains of Plasmodium falciparum

A 92,000 D protein was identified associated with the membrane of host erythrocytes infected with the FCB1 Plasmodium falciparum strain from Colombia. The same protein was identified in the knob-forming Gambian (and the Malayan Camp) strain, but was not present in all the corresponding knobless strains. In the FCB1 strain as well as in the FCR3 strain the protein is synthesized during the ring-stage period. The cleavage products of the 92,000 D protein were investigated by peptide mapping following limited proteolytic digestion with Staphylococcus aureus V8 protease. The 92,000 D protein cleavage products from both the Colombian and the Gambian strains were identical. Moreover, both the proteins were sensitive to trypsin and chymotrypsin and also to treatment with neuraminidase. Enzymatic removal of the protein from the erythrocyte membrane by trypsin or chymotrypsin did not affect parasite maturation. The merozoites thus produced were fully invasive and the morphology of the knobs was unaltered. When the erythrocyte membrane was treated with trypsin before the time of synthesis of the 92,000 D protein, it was not possible to identify the protein in membranes of later stages of infected erythrocytes, indicating that the protein cannot be inserted into the membrane cytoskeleton compartment. Knobs, however, were formed more or less normally, suggesting that it is not the accumulation of this protein which products the knobs.     

Protection of actin against proteolysis by complex formation with deoxyribonuclease I

G-actin bound to deoxyribonuclease I (DNase I) is resistant to digestion by trypsin and chymotrypsin. In the absence of DNase I, G-actin is cleaved by these proteases to yield a 33 500 molecular weight core protein which is not degraded further. The major sites of proteolytic action in the amino acid sequence of actin have been identified as being adjacent to residues arginine-62 and lysine-68 for trypsin and leucine-57 for chymotrypsin. These residues are rendered inaccessible to proteases in the buffer by complex formation with DNase I. Digestion of G-actin with pronase from Streptomyces griseus yields fragmentation patterns that are similar to those observed with trypsin and chymotrypsin. This is likely to be because the specificities of the major constituents of pronase resemble those of trypsin and chymotrypsin. Again, complex formation with DNase I protects the otherwise vulnerable bonds in actin against proteolysis. Incubation with subtilisin Carlsberg leads to complete digestion of G-actin. No subtilisin-resistant core protein accumulates during the incubation. Protection of G-actin when complexed to DNase I is less than complete in this case but still is significant. This is interpreted in terms of the broad specificity of subtilisin and the observed fragmentation pattern of free G-actin when treated with subtilisin.     

Evidence for an oxyanion hole in serine beta-lactamases and DD-peptidases.

A thionocephalosporin is shown to be a much poorer substrate of representative serine beta-lactamases of class A (RTEM-2) and class C (Enterobacter cloacae P99) and a much poorer inhibitor of the Streptomyces R61 DD-peptidase than is the analogous oxo beta-lactam. These results provide kinetic evidence for the existence of a catalytic oxyanion hole in these enzymes.     

Amino acid sequences around the cysteine residue of calf lens α-crystallin

1. Calf lens alpha-crystallin was carboxymethylated with radioactive sodium iodoacetate to label the thiol group. 2. The protein was then digested with trypsin or alternatively fractionated in urea to obtain the acidic (A) chains, which were then digested with trypsin. Either procedure gave two radioactive peptides containing carboxymethylcysteine. 3. These two peptides were closely related: the longer form contained 28 amino acid residues, and the shorter lacked two residues at the N-terminal end of the longer form. 4. The amino acid sequence of the peptides have been determined. 5. No evidence for the presence of more than one cysteine residue/chain was found. 6. The question of the molecular weight of the chains is discussed.     

Crystal structures of complexes between the R61 DD-peptidase and peptidoglycan-mimetic beta-lactams: a non-covalent complex with a "perfect penicillin"

The bacterial D-alanyl-D-alanine transpeptidases (DD-peptidases) are the killing targets of beta-lactams, the most important clinical defense against bacterial infections. However, due to the constant development of antibiotic-resistance mechanisms by bacteria, there is an ever-present need for new, more effective antimicrobial drugs. While enormous numbers of beta-lactam compounds have been tested for antibiotic activity in over 50 years of research, the success of a beta-lactam structure in terms of antibiotic activity remains unpredictable. Tipper and Strominger suggested long ago that beta-lactams inhibit DD-peptidases because they mimic the D-alanyl-D-alanine motif of the peptidoglycan substrate of these enzymes. They also predicted that beta-lactams having a peptidoglycan-mimetic side-chain might be better antibiotics than their non-specific counterparts, but decades of research have not provided any evidence for this. We have recently described two such novel beta-lactams. The first is a penicillin having the glycyl-L-alpha-amino-epsilon-pimelyl side-chain of Streptomyces strain R61 peptidoglycan, making it the "perfect penicillin" for this organism. The other is a cephalosporin with the same side-chain. Here, we describe the X-ray crystal structures of the perfect penicillin in non-covalent and covalent complexes with the Streptomyces R61 DD-peptidase. The structure of the non-covalent enzyme-inhibitor complex is the first such complex to be trapped crystallographically with a DD-peptidase. In addition, the covalent complex of the peptidyl-cephalosporin with the R61 DD-peptidase is described. Finally, two covalent complexes with the traditional beta-lactams benzylpenicillin and cephalosporin C were determined for comparison with the peptidyl beta-lactams. These structures, together with relevant kinetics data, support Tipper and Strominger's assertion that peptidoglycan-mimetic side-chains should improve beta-lactams as inhibitors of DD-peptidases.     

Development and applications of in-gel CNBr/tryptic digestion combined with mass spectrometry for the analysis of membrane proteins

Hydrophobic membrane proteins often have complex functions and are thus of great interest. However, their analysis presents a challenge because they are not readily soluble in polar solvents and often undergo aggregation. We present a sequential CNBr and trypsin in-gel digestion method combined with mass spectrometry for membrane protein analysis. CNBr selectively cleaves methionine residues. But due to the low number of methionines in proteins, CNBr cleavage produces a small number of large peptide fragments with MWs typically >2000, which are difficult to extract from gel pieces. To produce a larger number of smaller peptides than that obtained by using CNBr alone, we demonstrate that trypsin can be used to further digest the sample in gel. The use of n-octyl glucoside (n-OG) to enhance the digestion efficiency and peptide recovery was also studied. We demonstrate that the sensitivity of this membrane protein identification method is in the tens of picomole regime, which is compatible to the Coomassie staining gel-spot visualization method, and is more sensitive than other techniques reported in the literature. This CNBr/trypsin in-gel digestion method is also found to be very reproducible and has been successfully applied for the analysis of complex protein mixtures extracted from biological samples. The results are presented from a study of the analysis of bacteriorhodopsin, nitrate reductase 1 gamma chain, and a complex protein mixture extracted from the endoplasmic recticulum membrane of mouse liver.