Structure of a slow processing precursor penicillin acylase from Escherichia coli reveals the linker peptide blocking the active-site cleft

Penicillin G acylase is a periplasmic protein, cytoplasmically expressed as a precursor polypeptide comprising a signal sequence, the A and B chains of the mature enzyme (209 and 557 residues respectively) joined by a spacer peptide of 54 amino acid residues. The wild-type AB heterodimer is produced by proteolytic removal of this spacer in the periplasm. The first step in processing is believed to be autocatalytic hydrolysis of the peptide bond between the C-terminal residue of the spacer and the active-site serine residue at the N terminus of the B chain. We have determined the crystal structure of a slowly processing precursor mutant (Thr263Gly) of penicillin G acylase from Escherichia coli, which reveals that the spacer peptide blocks the entrance to the active-site cleft consistent with an autocatalytic mechanism of maturation. In this mutant precursor there is, however, an unexpected cleavage at a site four residues from the active-site serine residue. Analyses of the stereochemistry of the 260-261 bond seen to be cleaved in this precursor structure and of the 263-264 peptide bond have suggested factors that may govern the autocatalytic mechanism.     

Peptide substrate specificity of the membrane-bound metalloprotease of Leishmania

The promastigote surface protease (PSP) of Leishmania is a neutral membrane-bound zinc enzyme. The protease has no exopeptidase activity and does not cleave a large selection of substrates with chromogenic and fluorogenic leaving groups at the P1' site. The substrate specificity of the enzyme was studied by using natural and synthetic peptides of known amino acid sequence. The identification of 11 cleavage sites indicates that the enzyme preferentially cleaves peptides at the amino side when hydrophobic residues are in the P1' site and basic amino acid residues in the P2' and P3' sites. In addition, tyrosine residues are commonly found at the P1 site. Hydrolysis is not, however, restricted to these residues. These results have allowed the synthesis of a model peptide, H2N-L-I-A-Y-L-K-K-A-T-COOH, which is cleaved by PSP between the tyrosine and leucine residues with a kcat/Km ratio of 1.8 X 10(6) M-1 s-1. Furthermore, a synthetic nonapeptide overlapping the last four amino acids of the prosequence and the first five residues of mature PSP was found to be cleaved by the protease at the expected site to release the mature enzyme. This result suggests a possible autocatalytic mechanism for the activation of the protease. Finally, the hydroxamate-derivatized dipeptide Cbz-Tyr-Leu-NHOH was shown to inhibit PSP competitively with a KI of 17 microM.     

Gene cloning and molecular characterization of an extracellular poly(L-lactic acid) depolymerase from Amycolatopsis sp. strain K104-1

We have isolated a polylactide or poly(L-lactic acid) (PLA)-degrading bacterium, Amycolatopsis sp. strain K104-1, and purified PLA depolymerase (PLD) from the culture fluid of the bacterium. Here, we cloned and expressed the pld gene encoding PLD in Streptomyces lividans 1326 and characterized a recombinant PLD (rPLD) preparation. We also describe the processing mechanism from nascent PLD to mature PLD. The pld gene encodes PLD as a 24,225-Da polypeptide consisting of 238 amino acids. Biochemical and Western immunoblot analyses of PLD and its precursors revealed that PLD is synthesized as a precursor (prepro-type), requiring proteolytic cleavage of the N-terminal 35-amino-acid extension including the 26-amino-acid signal sequence and 9-residue prosequence to generate the mature enzyme of 20,904 Da. The cleavage of the prosequence was found to be autocatalytic. PLD showed about 45% similarity to many eukaryotic serine proteases. In addition, three amino acid residues, H57, D102, and S195 (chymotrypsin numbering), which are implicated in forming the catalytic triad necessary for cleavage of amide bond of substrates in eukaryotic serine proteases, were conserved in PLD as residues H74, D111, and S197. The G193 residue (chymotrypsin numbering), which is implicated in forming an oxyanion hole with residue S195 and forms an important hydrogen bond for interaction with the carbonyl group of the scissile peptide bond, was also conserved in PLD. The functional analysis of the PLD mutants H74A, D111A, and S197A revealed that residues H74, D111, and S197 are important for the depolymerase and caseinolytic activities of PLD and for cleavage of the prosequence from pro-type PLD to form the mature one. The PLD preparation had elastase activity which was not inhibited by 1 mM elastatinal, which is 10 times higher than needed for complete inhibition of porcine pancreatic elastase. The rPLD preparation degraded PLA with an average molecular mass of 220 kDa into lactic acid dimers through lactic acid oligomers and finally into lactic acid. The PLD preparation bound to high polymers of 3-hydoxybutyrate, epsilon-caprolacton, and butylene succinate as well as PLA, but it degraded only PLA.     

Rat procathepsin B. Proteolytic processing to the mature form in vitro.

Expression of rat procathepsin B in yeast led to the secretion of both the latent and mature forms of the enzyme. Culture in the presence of a cysteine proteinase inhibitor prevented this processing. We have expressed and purified a mutant form of rat procathepsin B whose active-site cysteine residue has been changed to a serine, and which also lacks the glycosylation site in the mature region of the protein. This non-active mutant protein was secreted essentially in an unprocessed form. The purified protein has been incubated with a variety of proteinases, and results indicate that cathepsins D and L, as well as mature cathepsin B itself, can produce a processed (single-chain) form of cathepsin B from this precursor. Amino-terminal sequencing of these processed forms has revealed that they are all elongated by a few residues with respect to the mature form found in vivo. The action of a combination of cathepsin B with dipeptidylpeptidase I produced a single-chain form of cathepsin B with the correct amino terminus. This work has also shown that the processing of procathepsin B to a single-chain form can be an autocatalytic process, in at least an intermolecular manner.     

Sequence of the Citrobacter freundii OS60 chromosomal ampC beta-lactamase gene

The Citrobacter freundii OS60 ampC beta-lactamase gene was sequenced and found to encode a 380-amino-acid-long precursor with a 19-residue signal peptide. The mature protein has a predicted molecular mass of 39781 Da. The first 60 residues of the purified enzyme, as determined by sequential Edman degradation, are identical to the amino acid sequence inferred from the gene sequence. Also, the amino acid composition determined for the purified beta-lactamase and that given by the gene sequence are in good agreement. 77% of the amino acid positions hold identical residues in the C. freundii and Escherichia coli K12 chromosomal AmpC beta-lactamases. This clearly puts the C. freundii enzyme into the class C of beta-lactamases. Of the 68 amino-terminal residues determined for the Enterobacter cloacae P99 beta-lactamase, 44 are identical to the corresponding residues of the C. freundii enzyme. All three enzymes, as well as that of Pseudomonas aeruginosa 18S/H are highly similar around the active-site serine at position 64 of the mature protein.     

Molecular cloning of the structural gene for alkaline elastase YaB, a new subtilisin produced by an alkalophilic Bacillus strain.

Alkaline elastase YaB is an extracellular serine protease of the alkalophilic Bacillus strain YaB. We cloned the structural gene, ale, and determined the nucleotide sequence. The mature enzyme (268 amino acids) was preceded by a putative signal sequence and a prosequence (27 and 83 amino acids, respectively). The mature enzyme was 55% homologous to subtilisin BPN'. Almost all the positively charged residues are predicted to be on the surface of the molecule, which would facilitate binding to elastin. The P1 substrate site-related sequences differed between alkaline elastase YaB and subtilisin BPN'.     

The crystal structure of a Cys25 -> Ala mutant of human procathepsin S elucidates enzyme-prosequence interactions

The crystal structure of the active-site mutant Cys25 --> Ala of glycosylated human procathepsin S is reported. It was determined by molecular replacement and refined to 2.1 Angstrom resolution, with an R-factor of 0.198. The overall structure is very similar to other cathepsin L-like zymogens of the C1A clan. The peptidase unit comprises two globular domains, and a small third domain is formed by the N-terminal part of the prosequence. It is anchored to the prosegment binding loop of the enzyme. Prosegment residues beyond the prodomain dock to the substrate binding cleft in a nonproductive orientation. Structural comparison with published data for mature cathepsin S revealed that procathepsin S residues Phe146, Phe70, and Phe211 adopt different orientations. Being part of the S1' and S2 pockets, they may contribute to the selectivity of ligand binding. Regarding the prosequence, length, orientation and anchoring of helix alpha3p differ from related zymogens, thereby possibly contributing to the specificity of propeptide-enzyme interaction in the papain family. The discussion focuses on the functional importance of the most conserved residues in the prosequence for structural integrity, inhibition and folding assistance, considering scanning mutagenesis data published for procathepsin S and for its isolated propeptide.     

Characterization of a cloned subtilisin-like serine proteinase from a psychrotrophic Vibrio species.

The gene encoding a subtilisin-like serine proteinase in the psychrotrophic Vibrio sp. PA44 has been successfully cloned, sequenced and expressed in Escherichia coli. The gene is 1593 basepairs and encodes a precursor protein of 530 amino acid residues with a calculated molecular mass of 55.7 kDa. The enzyme is isolated, however, as an active 40.6-kDa proteinase, without a 139 amino acid residue N-terminal prosequence. Under mild conditions the enzyme undergoes a further autocatalytic cleavage to give a 29.7-kDa proteinase that retains full enzymatic activity. The deduced amino acid sequence of the enzyme has high homology to proteinases of the proteinase K family of subtilisin-like proteinases. With respect to the enzyme characteristics compared in this study the properties of the wild-type and recombinant proteinases are the same. Sequence analysis revealed that especially with respect to the thermophilic homologues, aqualysin I from Thermus aquaticus and a proteinase from Thermus strain Rt41A, the cold-adapted Vibrio-proteinase has a higher content of polar/uncharged amino acids, as well as aspartate residues. The thermophilic enzymes had a higher content of arginines, and relatively higher number of hydrophobic amino acids and a higher aliphatic index. These factors may contribute to the adaptation of these proteinases to different temperature conditions.     

Structure of a baculovirus sulfhydryl oxidase, a highly divergent member of the erv flavoenzyme family

Genomes of nucleocytoplasmic large DNA viruses (NCLDVs) encode enzymes that catalyze the formation of disulfide bonds between cysteine amino acid residues in proteins, a function essential for the proper assembly and propagation of NCLDV virions. Recently, a catalyst of disulfide formation was identified in baculoviruses, a group of large double-stranded DNA viruses considered phylogenetically distinct from NCLDVs. The NCLDV and baculovirus disulfide catalysts are flavin adenine dinucleotide (FAD)-binding sulfhydryl oxidases related to the cellular Erv enzyme family, but the baculovirus enzyme, the product of the Ac92 gene in Autographa californica multiple nucleopolyhedrovirus (AcMNPV), is highly divergent at the amino acid sequence level. The crystal structure of the Ac92 protein presented here shows a configuration of the active-site cysteine residues and bound cofactor similar to that observed in other Erv sulfhydryl oxidases. However, Ac92 has a complex quaternary structural arrangement not previously seen in cellular or viral enzymes of this family. This novel assembly comprises a dimer of pseudodimers with a striking 40-degree kink in the interface helix between subunits. The diversification of the Erv sulfhydryl oxidase enzymes in large double-stranded DNA viruses exemplifies the extreme degree to which these viruses can push the boundaries of protein family folds.     

Extracellular secretion of Escherichia coli heat-stable enterotoxin I across the outer membrane.

Escherichia coli heat-stable enterotoxin Ip (STIp) is an extracellular toxin consisting of 18 amino acid residues that is synthesized as a precursor of pre (amino acid residues 1 to 19), pro (amino acid residues 20 to 54), and mature (amino acid residues 55 to 72) regions. The precursor synthesized in the cytoplasm is translocated across the inner membrane by the general export pathway consisting of Sec proteins. The pre region functions as a leader peptide and is cleaved during translocation. However, it remains unknown how the resulting peptide (pro-mature peptide) translocates across the outer membrane. In this study, we investigated the structure of the STIp that passes through the outer membrane to determine how it translocates through the outer membrane. The results showed that the pro region is cleaved in the periplasmic space. The generated peptide becomes the mature form of STIp, which happens to have disulfide bonds, which then passes through the outer membrane. We also showed that STIp with a carboxy-terminal peptide consisting of 3 amino acid residues passes through the outer membrane, whereas STIp with a peptide composed of 37 residues does not. Amino acid analysis of mutant STIp purified from culture supernatant revealed that the peptide composed of 37 amino acid residues was cleaved into fragments of 5 amino acid residues. In addition, analyses of STIps with a mutation at the cysteine residue and the dsbA mutant strain revealed that the formation of an intramolecular disulfide bond within STIp is not absolutely required for the mature region of STIp to pass through the outer membrane.     

Mutational analysis of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase propeptide processing

Glutamine phosphoribosylpyrophosphate amidotransferase from Bacillus subtilis is a member of an N-terminal nucleophile hydrolase enzyme superfamily, several of which undergo autocatalytic propeptide processing to generate the mature active enzyme. A series of mutations was analyzed to determine whether amino acid residues required for catalysis are also used for propeptide processing. Propeptide cleavage was strongly inhibited by replacement of the cysteine nucleophile and two residues of an oxyanion hole that are required for glutaminase function. However, significant propeptide processing was retained in a deletion mutant with multiple defects in catalysis that was devoid of enzyme activity. Intermolecular processing of noncleaved mutant enzyme subunits by active wild-type enzyme subunits was not detected in hetero-oligomers obtained from a coexpression experiment. While direct in vitro evidence for autocatalytic propeptide cleavage was not obtained, the results indicate that some but not all of the amino acid residues that have a role in catalysis are also needed for propeptide processing.     

Activation of the proteinase B precursor of the yeast Saccharomyces cerevisiae by autocatalysis and by an internal sequence.

Proteinase B (PrB) is a subtilisin-like serine protease found in the vacuole of the yeast Saccharomyces cerevisiae. It is first made as a large precursor that consists of a putative signal sequence, a 260-amino acid pro region, the serine protease domain, and two small COOH-terminal post regions (Moehle, C. M., Dixon, C. K., and Jones, E. W. (1989) J. Cell Biol. 108, 309-324). This precursor is glycosylated and proteolytically processed at least three times before mature enzyme is formed. To determine whether an intact PrB catalytic site is required for proteolytic processing of the precursor, point mutations were generated at the codons for the active site serine or aspartate residues by site-directed mutagenesis. The effect of these mutations on PrB processing suggests that the large pro region may be cleaved by an intramolecular, autocatalytic mechanism. The properties of a prb1 mutant that accumulates a 37-kDa precursor in addition to mature sized mutant PrB antigen suggests that the final proteolytic cleavage step is also autocatalytic. A prb1 deletion that lacks codons for the large pro region was made to test whether this part of the precursor is required for formation of mature PrB. Analysis of this mutant revealed two functions for this region: it prevents N-linked glycosylation of the serine protease domain and it allows the PrB precursor to be processed by proteinase A. The pro region can fulfill this latter function if added as a separate molecule, so long as glycosylation of the catalytic domain is prevented by other means.     

Isolation, characterization and molecular cloning of a lipolytic enzyme secreted from Malassezia pachydermatis

Lipophilic Malassezia species may induce catheter-associated sepsis in premature neonates and immunocompromised patients receiving parenteral lipid emulsions. To assess the participation of lipolytic enzymes in the pathogenesis of this yeast, we cloned a gene encoding the enzyme. A lipolytic enzyme in the culture supernatant of Malassezia pachydermatis was purified 210-fold to homogeneity. The enzyme showed high esterase activity toward p-nitrophenyl octanoate. The cDNA encoding the enzyme was cloned using a degenerate oligonucleotide primer constructed from the N-terminal amino acid sequence. The cDNA consisted of 1582 bp, including an open reading frame encoding 470 amino acids. The first 19 amino acids and the following 13 amino-acid sequence were predicted to be the signal peptides for secretion and prosequence, respectively. The predicted molecular mass of the 438-amino acid mature protein was 48 kDa. Analysis of the deduced amino acid sequence revealed that it contains the consensus motif (Gly-X-Ser-X-Gly), which is conserved among lipolytic enzymes. Homology investigations showed that the enzyme has similarities principally with 11 lipases produced by Candida albicans (29-34% identity) and some other yeast lipases.     

Cloning, sequence analysis and expression of a gene encoding an organic solvent- and detergent-tolerant cholesterol oxidase of Burkholderia cepacia strain ST-200

Burkholderia cepacia strain ST-200 produces an extracellular cholesterol oxidase which is stable and highly active in the presence of organic solvents. This cholesterol oxidase produces 6beta-hydroperoxycholest-4-en-3-one from cholesterol, with the consumption of two moles of O2 and the formation of one mole of H2O2. The structural gene encoding the cholesterol oxidase was cloned and sequenced. The primary translation product was predicted to be 582 amino acid residues. The mature product is composed of 539 amino acid residues and is preceded by a signal sequence of 43 residues. The cloned gene was expressed as an active product in Escherichia coli and the product was localized in the periplasmic space. The cholesterol oxidase produced from E. coli was purified to homogeneity from the periplasmic fraction. The purified enzyme was highly stable in the presence of various organic solvents or detergents, as compared with the commercially available cholesterol oxidases tested.     

Brain 4-aminobutyrate aminotransferase. Isolation and sequence of a cDNA encoding the enzyme.

4-Aminobutyrate aminotransferase is a key enzyme of the 4-aminobutyric acid shunt. It is responsible for the conversion of the neurotransmitter 4-aminobutyrate to succinic semialdehyde. By using oligonucleotide probes based on partial amino acid sequence data for the pig brain enzyme, several overlapping cDNA clones of 2.0-2.2 kilobases in length have been isolated. The largest cDNA clone was selected for sequence analysis. The amino acid sequence predicted from the cDNA sequence shows that the precursor of 4-aminobutyrate aminotransferase consists of the mature enzyme of 473 amino acid residues and an amino-terminal segment of 27 amino acids attributed to the signal peptide. The cofactor pyridoxal-5-P is bound to lysine residue 330 of the deduced amino acid sequence of the mature enzyme.     

Structural basis of proteolytic activation of L-phenylalanine oxidase from Pseudomonas sp. P-501

The mature form of l-phenylalanine oxidase (PAOpt) from Pseudomonas sp. P-501 was generated and activated by the proteolytic cleavage of a noncatalytic proenzyme (proPAO). The crystal structures of proPAO, PAOpt, and the PAOpt-o-amino benzoate (AB) complex were determined at 1.7, 1.25, and 1.35A resolutions, respectively. The structure of proPAO suggests that the prosequence peptide of proPAO occupies the funnel (pathway) of the substrate amino acid from the outside of the protein to the interior flavin ring, whereas the funnel is closed with the hydrophobic residues at its vestibule in both PAOpt and the PAOpt-AB complex. All three structures have an oxygen channel that is open to the surface of the protein from the flavin ring. These results suggest that structural changes were induced by proteolysis; that is, the proteolysis of proPAO removes the prosequence and closes the funnel to keep the active site hydrophobic but keeps the oxygen channel open. The possibility that an interaction of the hydrophobic side chain of substrate with the residues of the vestibule region may open the funnel as a putative amino acid channel is discussed.     

Cloning and nucleotide sequence of cDNA for the plastid glycerol-3-phosphate acyltransferase from squash

The partial amino acid sequence and amino acid composition of acyl-(acyl-carrier-protein):glycerol-3-phosphate acyltransferase purified from squash cotyledons were determined. cDNAs encoding this enzyme were isolated from lambda gt 11 cDNA libraries made from poly(A)+ RNA of squash cotyledons by immunological selection and cross-hybridization. One of the resultant clones contained a cDNA insert of 1426 base pairs and an open reading frame of 1188 base pairs. The amino acid sequence deduced from the nucleotide sequence matched the partial amino acid sequence determined for the enzyme. The results suggest that a precursor protein of 396 amino acid residues is processed to the mature enzyme of 368 amino acid residues, losing a leader peptide of 28 amino acid residues. Relative molecular masses of the precursor and mature proteins were calculated to be 43,838 and 40,929 Da, respectively.     

Cloning and sequence of cDNA encoding a peptide C-terminal alpha-amidating enzyme from Xenopus laevis

The C-terminal alpha-amide formation of the peptides is one of the most important events of prohormone processing. We have recently isolated an alpha-amidating enzyme, AE-I, from Xenopus laevis skin, which is the only enzyme ever purified to homogeneity. In this study, we report cloning and sequence of cDNA encoding AE-I. Our results indicate that enzyme AE-I is initially synthesized as a precursor with 400 amino acid residues, which is further processed to the mature enzyme consisting of 344 residues. Preliminary expression in E. coli of the cDNA corresponding to AE-I was found to produce an enzyme with appreciable alpha-amidating activity.     

Asparaginyl endopeptidase (VmPE-1) and autocatalytic processing synergistically activate the vacuolar cysteine proteinase (SH-EP).

A vacuolar cysteine proteinase, designated SH-EP, is synthesized in cotyledons of germinated Vigna mungo seeds and is responsible for degradation of the seed proteins accumulated in protein bodies (protein storage vacuoles). SH-EP belongs to the papain proteinase family and has a large N-terminal prosegment consisting of 104 amino acid residues and a C-terminal prosegment of 10 amino acid residues. It has been suggested that an asparaginyl endopeptidase, V. mungo processing enzyme 1 (VmPE-1), is involved in the N-terminal post-translational processing of SH-EP. The recombinant proform of SH-EP (rSH-EP) was produced in Escherichia coli cells, purified to homogeneity and refolded by stepwise dialysis. 31P-NMR analysis of intact germinated cotyledons revealed that the vacuolar pH of cotyledonary cells changes from 6.04 to 5.47 during seed germination and early seedling growth. rSH-EP was converted in vitro to the mature form through autocatalytic processing at a pH mimicking the vacuolar pH at the mid and late stages of seed germination, but not at the pH of the early stage. VmPE-1 accelerated the rate of processing of rSH-EP in vitro at the pH equivalent to the vacuolar pH at the early and mid stages of germination. In addition, the cleavage sites of the in vitro processed intermediates and the mature form of SH-EP were identical to those of SH-EP purified from germinated cotyledons of V. mungo. We propose that the asparaginyl endopeptidase (VmPE-1)-mediated processing mainly functions in the activation of proSH-EP at the early stage of seed germination, and both VmPE-1-mediated and autocatalytic processings function synergistically in the activation of proSH-EP in cotyledons at the mid and late stages.     

Structural and catalytic properties of copper in lysyl oxidase

The spectral and catalytic properties of the copper cofactor in highly purified bovine aortic lysyl oxidase have been examined. As isolated, various preparations of purified lysyl oxidase are associated with 5-9 loosely bound copper atoms per molecule of enzyme which are removed by dialysis against EDTA. The enzyme also contains 0.99 +/- 0.10 g atom of tightly bound copper per 32-kDa monomer which is not removed by this treatment. The copper-free apoenzyme, prepared by dialysis of lysyl oxidase against alpha,alpha'-dipyridyl in 6 M urea, catalyzed neither the oxidative turnover of amine substrates nor the anaerobic production of aldehyde at levels stoichiometric with enzyme active site content, thus contrasting with the ping pong metalloenzyme. Moreover, the spectrum of the apoenzyme was not measurably perturbed upon anaerobic incubation with n-butylamine, while difference absorption bands were generated at 250 and 308 nm in the spectrum of the metalloenzyme incubated under the same conditions. A difference absorption band also developed at 300-310 nm upon anaerobic incubation of pyrroloquinoline quinone, the putative carbonyl cofactor of lysyl oxidase, with n-butylamine. Full restoration of catalytic activity occurred upon the reconstitution of the apoenzyme with 1 g atom of copper/32-kDa monomer, whereas identical treatment of the apoenzyme with divalent salts of zinc, cobalt, iron, mercury, magnesium, or cadmium failed to restore catalytic activity. The EPR spectrum of copper in lysyl oxidase is typical of the tetragonally distorted, octahedrally coordinated Cu(II) sites observed in other amine oxidases and indicates coordination by at least three nitrogen ligands. The single copper atom in the lysyl oxidase monomer is thus essential at least for the catalytic and possibly for the structural integrity of this protein.