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Query: UNIPROT:P06889 (Mol)
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Globular proteins can be denatured by changing pH and ionic strength. Much recent evidence has led to the surprising conclusion that there are two acid-denatured states: one highly unfolded and the other more compact, sometimes called the "molten globule." Here we describe a molecular theory for electrostatic stability of globular proteins based on the properties of the constituent amino acids: oil/water partition coefficients, pK values of the titratable groups, and their temperature dependences. Predicted denaturation temperatures vs. pH are in good agreement with experiments of other workers on myoglobin. The theory also predicts two populations of denatured species, one open and the other more compact, with densities in the range found experimentally for molten globular states. In addition, it predicts a phase diagram (stability vs. pH, ionic strength) in good agreement with experiments of Goto and Fink [Goto, Y. & Fink, A. L. (1989) Biochemistry 28, 945-952; and Goto, Y. & Fink, A. L. (1990) J. Mol. Biol. 214, 803-805]. The well-known salt destabilization of myoglobin has been generally considered evidence for ion pairing, but the present theory, based on smeared charge repulsion, explains the salt destabilization at low pH without ion pairing. In addition, for myoglobin the theory predicts salt stabilization at high pH, as observed for beta-lactamase by Goto and Fink.
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PMID:Protein stability: electrostatics and compact denatured states. 203 62

Production of beta-lactamases, and of the plasmid-encoded TEM- and SHV-type enzymes in particular, is the most common mechanism of resistance against beta-lactam antibiotics in Gram-negative bacteria. The two ubiquitous types of enzyme have a large spectrum of activity and preferentially hydrolyse the penicillins as well as some first- and second-generation cephalosporins. Recently, point mutations in the corresponding genes have been observed, apparently selected for, in the clinical setting, by originally 'beta-lactamase-stable' third-generation cephalosporins or by monobactams, which fall into the substrate range of the mutant or 'extended-spectrum' beta-lactamases. The point mutations are clustered in three areas, each adjacent to one of the seven evolutionarily conserved boxes described by Joris et al. (1988). The substituted amino acids at positions 102 (adjacent to the alpha-3 helix), 162 (adjacent to the alpha-7 helix) and 235, 236 and 237 (on the beta-3 strand) are located in close proximity to the active-site cavity and are thought to open up novel enzyme-substrate interactions, involving, in particular, the oxyimino moieties of the newer beta-lactam compounds.
Mol Microbiol 1990 Oct
PMID:Molecular evolution of ubiquitous beta-lactamases towards extended-spectrum enzymes active against newer beta-lactam antibiotics. 207 52

The enzyme TEM beta-lactamase constitutes a versatile gene-fusion marker for studies on membrane proteins and protein export in bacteria. The mature form of this normally periplasmic enzyme displays readily detectable and distinctly different phenotypes when localized to the bacterial cytoplasm versus the periplasm, and thus provides a useful alternative to alkaline phosphatase for probing the topology of cytoplasmic membrane proteins. Cells producing translocated forms of beta-lactamase can be directly selected as ampicillin-resistant colonies, and consequently a beta-lactamase fusion approach can be used for positive selection for export signals, and for rapid assessment of whether any protein expressed in Escherichia coli inserts into the bacterial cytoplasmic membrane. The level of ampicillin resistance conferred on a cell by an extracytoplasmic beta-lactamase derivative depends on its level of expression, and therefore a beta-lactamase fusion approach can be used to directly select for increased yields of any periplasmic or membrane-bound gene products expressed in E. coli.
Mol Microbiol 1990 Oct
PMID:Beta-lactamase as a probe of membrane protein assembly and protein export. 207 55

The beta-lactam antibiotic cloxacillin can inhibit secretion of prokaryotic lipo-beta-lactamase into the periplasm of yeast. The results indicate that this phenomenon is specific with respect to both the antibiotic and the lipo-beta-lactamase whose secretion is affected, strongly suggesting that this involves an interaction between the enzyme and its substrates. The effect of the antibiotic on secretion is reversible. With different beta-lactam antibiotics, the clearest difference is observed between type A and type S penicillins; the former exert a strong inhibition of secretion whereas the latter exhibit a weak effect or no effect at all. Type A penicillins have been previously shown to cause a conformational change in various beta-lactamases. Mature lipo-beta-lactamase species in yeast were localized either to the periplasmic space or bound to the outer surface of the cytoplasmic membrane and thus exposed to periplasm. The results are consistent with the hypothesis that binding of cloxacillin to lipo-beta-lactamase induces a conformation on the protein that is unfavourable for its release from the membrane.
Mol Microbiol 1990 Dec
PMID:Specific beta-lactam antibiotics inhibit secretion of lipo-beta-lactamase in yeast. 208 28

An out-of-frame fusion between the penicillinase gene (penP) of Bacillus licheniformis and the beta-galactosidase gene (lacZ) of Escherichia coli was shown to direct the synthesis of an active beta-galactosidase with the same electrophoretic mobility as the wild-type protein, both in B. subtilis and E. coli. This synthesis was dependent on translation of the truncated penP gene and appeared to result from translational coupling. The fusion point between penP and lacZ contained the sequence AUAG, in which the UAG and AUA codons were in-frame with the penP and lacZ reading units, respectively. N-terminal amino acid sequence analysis of the beta-galactosidase protein suggested that, both in B. subtilis and E. coli, reinitiation of translation occurred at the AUA codon present at the gene fusion point.
Mol Gen Genet 1990 Apr
PMID:Translational coupling in a penP-lacZ gene fusion in Bacillus subtilis and Escherichia coli: use of AUA as a restart codon. 211 12

Non-beta-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae (CMRNG strains) produce altered forms of penicillin-binding protein 2 (PBP2) that have decreased affinity for penicillin. A feature of PBP2 from all CMRNG strains is the presence of an additional residue (Asp-345A) that is absent from PBP2 of penicillin-sensitive strains. The role of the additional aspartic acid residue in the decreased affinity of PBP2 is unclear as PBP2 of all previously examined CMRNG strains possess several other amino acid sequence alterations, in addition to the insertion of Asp-345A, compared to PBP2 of penicillin-sensitive strains. Site-directed mutagenesis has been used to insert the Asp-345A codon into the penA gene from a penicillin-sensitive gonococcus. The resulting penA gene expressed an altered form of PBP2 that had a decreased affinity for benzylpenicillin and was able to transform a penicillin-sensitive strain of N. gonorrhoeae to an increased level of resistance to benzylpenicillin. Insertion of amino acids other than aspartic acid did not produce forms of PBP2 that provided increased resistance to penicillin. Removal of the Asp-345A codon from the penA gene of a CMRNG strain reduced its ability to transform a penicillin-sensitive strain to an increased level of penicillin resistance. The reduction in the affinity of PBP2 in CMRNG strains is therefore largely, although not exclusively, due to the insertion of Asp-345A. Clinical isolates that produce altered forms of PBP2 that differ from that of penicillin-sensitive strains only in the insertion of Asp-345A have been identified.
Mol Microbiol 1990 Jun
PMID:Insertion of an extra amino acid is the main cause of the low affinity of penicillin-binding protein 2 in penicillin-resistant strains of Neisseria gonorrhoeae. 212 May 42

The Escherichia coli rap mutant inhibits vegetative growth of bacteriophage lambda. Phage mutations termed bar, which overcome the rap defect, have been mapped to three genetic loci in the pL operon. Plasmids with a lambda wild-type bar DNA segment cloned downstream from an active promoter cannot be maintained in rap mutant bacteria. The viability of a rap mutant strain decreases rapidly after induction of transcription through bar regions present on plasmids. Under these (restrictive) conditions the expression of plasmid-encoded beta-lactamase and plasmid DNA replication are arrested, but plasmid RNA synthesis continues for several hours. Analysis of protein extracts from E. coli rap cells containing bar plasmids revealed that both plasmid and bacterial protein synthesis are inhibited under restrictive conditions. In addition, unlike other RNAs tested, the chemical half-life of bar RNA increases 3.5-fold relative to the half-life of bar RNA under permissive conditions. We propose that transcription through the bar region, or the accumulation of bar RNA, results in an irreversible defect in cellular mRNA translation. This defect eventually kills the rap cells, and thus prevents bar plasmid maintenance.
J Mol Biol 1990 Nov 20
PMID:A short DNA sequence from lambda phage inhibits protein synthesis in Escherichia coli rap. 214 20

Tn552, one of several closely related beta-lactamase-encoding transposons from Staphylococcus aureus, has a novel set of putative transposition functions. Each is homologous with a well-characterized function from a different type of mobile genetic element. Thus, Tn552 encodes: (i) resL-binL, a co-integrate resolution system homologous with those of Tn3 family elements; (ii) p480, a potential transposase significantly homologous with the DNA integrases of eukaryotic retroviruses and retrotransposons; and (iii) p271, a potential ATP-binding protein that shows homology with the B protein of phage Mu. The 3' terminal nucleotides of Tn552 (CA), adjacent to which p480 might cleave, are the same as those of retroviruses, retrotransposons and phage Mu. The presumptive resolvase (BinL) is very closely related to BinR, which was identified as a DNA invertase and is now shown to resolve an artificial co-integrate in vivo. Furthermore, the structure of the derivative of Tn552 found in the staphylococcal plasmid pI258 can be explained by a BinL (or BinR)-mediated site-specific deletion ('resolution') event. Thus, pI258 contains only the right-hand half of Tn552, which encodes the beta-lactamase and two regulatory proteins. The latter are homologous with the beta-lactamase gene repressor and co-inducer of Bacillus licheniformis. Interestingly, the order of the regulatory genes is reversed in S. aureus compared with Bacillus licheniformis.
Mol Microbiol 1990 Jun
PMID:Tn552, a novel transposable element from Staphylococcus aureus. 217 Aug 15

Expression of mutant ompA genes, encoding the 325 residue Escherichia coli outer membrane protein OmpA, caused an inhibition of synthesis of the structurally unrelated outer membrane porins OmpC and OmpF and of wild-type OmpA, but not of the periplasmic beta-lactamase. There was no accumulation of precursors of the target proteins and the inhibitory mechanism operated at the level of translation. So far only alterations around residue 45 of OmpA have been found to affect this phenomenon. Linkers were inserted between the codons for residues 45 and 46. A correlation between size and sequence of the resulting proteins and presence or absence of the inhibitory effect was not found, indicating that the added residues acted indirectly by altering the conformation of other parts of the mutant OmpA. To be effective, the altered polypeptides had to be channelled into the export pathway. Internal deletions in effector proteins, preventing incorporation into the membrane, abolished effector activity. The results suggest the existence of a periplasmic component that binds to OmpA prior to membrane assembly; impaired release of this factor from mutant OmpA proteins may trigger inhibition of translation. The factor could be a See B-type protein, keeping outer membrane proteins in a form compatible with membrane assembly.
J Mol Biol 1990 Nov 05
PMID:Export of altered forms of an Escherichia coli K-12 outer membrane protein (OmpA) can inhibit synthesis of unrelated outer membrane proteins. 217 52

The promoter region of the gene encoding the extracellular DD-peptidase/penicillin-binding protein of Streptomyces R61 has been identified by in vivo promoter probing and S1 mapping. A secretion vector, pDML116, was constructed by inserting into the multicopy Streptomyces plasmid pIJ702, a 247 bp DNA sequence that contained the transcriptional, translational and secretory signals and the 12 amino acid N-terminal region-encoding sequence of the mature Streptomyces DD-peptidase/penicillin-binding protein. Insertion, downstream of this 247 bp segment, of the Streptomyces R61 DD-peptidase-encoding gene or the Escherichia coli R-TEM beta-lactamase-encoding gene yielded plasmids pDML120 and pDML128, respectively, which allowed expression and secretion of the relevant enzymes by Streptomyces lividans. The maximal secretion levels obtained were 42 mg protein/ml for the autologous Streptomyces DD-peptidase and 0.9 mg protein/ml for the heterologous E. coli beta-lactamase.
Mol Gen Genet 1990 Aug
PMID:Transcriptional analysis of the DD-peptidase/penicillin-binding protein-encoding dac gene of Streptomyces R61: use of the promoter and signal sequences in a secretion vector. 217 84


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