The role of the non-conserved residue at position 104 of class A beta-lactamases in susceptibility to mechanism-based inhibitors.

The role of the non-conserved amino acid residue at position 104 of the class A beta-lactamases, which comprises a highly conserved sequence of amino acids at the active sites of these enzymes, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace Asp104 with the corresponding Staphylococcus aureus PC1 residue Ala104. Kinetic data obtained with the purified Asp104Ala B. cereus 569/H beta-lactamase I was compared to that obtained from the wild-type B. cereus and S. aureus enzymes. Replacement of amino acid residue 104 had little effect on the Michaelis parameters for the hydrolysis of both S- and A-type penicillins. Relative to wild-type enzyme, the Asp104Ala beta-lactamase I had 2-fold higher Km values for benzylpenicillin and methicillin, but negligible difference in Km for ampicillin and oxacillin. However, kcat values were also slightly increased resulting in little change in catalytic efficiency, kcat/Km. In contrast, the Asp104Ala beta-lactamase I became more like the S. aureus enzyme in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amido-penicillanic acid sulfone with respect to both response to the inhibitors and subsequent enzymatic properties. Based on the known three-dimensional structures of the Bacillus licheniformis 749/C, Escherichia coli TEM and S. aureus PC1 beta-lactamases, a model for the role of the non-conserved residue at position 104 in the process of inactivation by mechanism-based inhibitors is proposed.

Lysine: N6-hydroxylase: stability and interaction with ligands.

Recombinant lysine:N6-hydroxylase, rIucD, which is isolated as an apoenzyme, requires FAD and NADPH for its catalytic function. rIucD preparations have been found to undergo time-dependent loss in monooxygenase function due to aggregation from the initial tetrameric state to a polytetrameric form(s), a process which is reversible by treatment with thiols. Ligand-induced conformational changes in rIucD were assessed by monitoring its CD spectra, DSC profile, and susceptibility to both endo- as well as exopeptidases. The first two methods indicated the absence of any significant conformational change in rIucD, while the last approach revealed that FAD, and its analog ADP, can protect the protein from the deleterious action of proteases. NADPH was partially effective and L-lysine was ineffective in this regard. Deletion of the C-terminal segment, either by treatment with carboxypeptidase Y or by mutagenesis of iucD, results in the loss of rIucD's monooxygenase activity. These findings demonstrate the crucial role of the C-terminal segment in maintaining rIucD in its native conformation.

Effect of selective cysteine --> alanine replacements on the catalytic functions of lysine: N6-hydroxylase.

Recombinant lysine: N6-hydroxylase, rIucD, catalyzes the conversion of L-lysine to its N6-hydroxy derivative. Re-examination of the nucleotide sequence of iucD, the gene encoding for the enzyme, has revealed a few discrepancies in the data documented in literature and the corrected version is presented. The revised nucleotide sequence predicts the presence of five cysteine residues in the primary structure of IucD. Two of these residues, cysteine 51 and cysteine 158 are alkylatable by iodoacetate in the native conformation of the protein resulting in a loss of monooxygenase activity while their replacement with alanine has no such adverse effect. Site directed mutagenesis studies have enabled an assessment of the reactivity of these cysteine residue(s) towards thiol modifying agents.

The role of the nonconserved residues at position 167 of class A beta-lactamases in susceptibility to mechanism-based inhibitors.

Differences in specificities between the class A beta-lactamases for both substrate and inhibitors are known. The role of the nonconserved amino acid residue at position 167 of the class A enzyme, which forms a cis bond with the catalytically essential Glu-166 residue, in both the hydrolysis of beta-lactam substrates and inactivation by mechanism-based inhibitors, was investigated. Site-directed mutagenesis was performed on the penPC gene encoding the Bacillus cereus 569/H beta-lactamase I to replace thr-167 with the corresponding Staphylococcus aureus PC1 residue Ile. Kinetic data obtained from the purified Thr-167-Ile B. cereus 569/H beta-lactamase was compared to that obtained from the wild-type B. cereus and S. aureus enzymes and indicated that the replacement had little effect on the Michaelis parameters for the hydrolysis of S- and A-type penicillins. However, the Thr-167-Ile enzymes became more S. aureus PC1-like in its response to the mechanism-based inhibitors clavulanic acid and 6-beta-(trifluoromethane sulfonyl)amidopenicillanic acid sulfone. A model for the role of this nonconserved residue at position 167 in the mechanism of inactivation by mechanism-based inhibitors is proposed.

Physico-chemical characterization of a recombinant cytoplasmic form of lysine: N6-hydroxylase.

A recombinant cytoplasmic preparation of lysine: N6-hydroxylase, IucD398, with a deletion of 47 amino acids at the N-terminus, was purified to homogeneity. IucD398 is capable of N-hydroxylation of L-lysine upon supplementation with FAD and NADPH. The enzyme is stringently specific with L-lysine and (S)-2-aminoethyl-L-cysteine serving as substrates. Protonophores, FCCP and CCCP, as well as cinnamylidene, have been found to serve as potent inhibitors of lysine: N6-hydroxylation by virtue of their ability to interfere in the reduction of the flavin cofactor.

Breaching the conformational integrity of the catalytic triad of the serine protease plasmin: localized disruption of a side chain of His-603 strongly inhibits the amidolytic activity of human plasmin.

Site-directed mutagenesis has been used to construct a cDNA that encodes a recombinant variant human plasminogen (hPg) containing a Pro-611-->Ile mutation (MrhPg). The mutein was expressed in recombinant baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE), and purified. After activation of this zymogen to its corresponding form of the serine protease plasmin (MrhPm), this latter enzyme was essentially inactive toward an amide plasmin substrate, most likely from alteration of the spatial relationships of the active-site His-603 to its partners of the catalytic triad, Asp-646 and Ser-741. Partial amidolytic activity of MrhPm was restored as a consequence of imidazole addition to the assay medium, due to an increase in the catalytic constant kcat of the enzyme. The serine protease inhibitor, diisopropylphosphofluoridate, when preincubated with MrhPm, did not inhibit restoration of its amidolytic activity with imidazole, whereas diisopropylphosphofluoridate did inhibit the amidolytic activity of MrhPm in the presence of imidazole. This result implies that His-603 directly influences the nucleophilic character of Ser-741. When imidazole as pretreated with alpha-N-tosyl-L-lysine chloromethyl ketone, the ability of this imidazole solution to restore amidolytic activity to MrhPm was eliminated, suggesting that N alpha-(p-tosyl)lysine chloromethyl ketone directs into the binding pocket a derivatized form of imidazole, which is ineffective as an His-603 substitute. These results indicate that the conformational reorientation of His-603 results in a malfunctional catalytic triad in the serine protease MrhPm, thus leading to an inactive enzyme despite the presence of all three essential amino acids of the catalytic triad. Addition of extramolecular imidazole restores a portion of the amidolytic activity of this mutant enzyme. These data also argue for an enzyme mechanism in which the active-center His-603 residue directly influences the nucleophilicity of the active-site Ser 741 residue.

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster.

The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)

A novel role for calcite in calcium homeostasis.

Calcium carbonate (CaCO3) minerals are known to be deposited in a wide array of different organisms, ranging from microbes to vertebrates [(1989) On Biomineralization, Oxford University Press, New York]. Calcite, aragonite and vaterite are the major crystalline structural polymorphs of CaCO3 associated with living systems, and participate in a variety of biological functions [(1989) Biomineralization: Chemical and Biochemical Perspectives, VCH Publishers, Weinham, Germany; (1991) Advances in Inorganic Chemistry 36, 137-200]. Here we report on the ability of a soil bacterium to synthesize calcite in a calcium-stressed environment. The elaboration of this exocellular crystalline residue enables the organism to regulate its calcium content. The attainment of calcium homeostasis via the exocellular deposition of bacterial calcite with unique crystal habits is a novel biological phenomenon.

Flavobacterium meningosepticum peptide:N-glycosidase: influence of ionic strength on enzymatic activity.

Flavobacterium meningosepticum peptide:N-glycosidase-mediated deglycosylation of N-linked glycan strands of glycoproteins has been found to be strongly influenced by the ionic strength of the assay medium. By use of a modification of a previously published assay procedure for quantitative analysis of glycan release we have been able to improve reproducibility and thus to compare the extent of deglycosylation achieved under a variety of conditions of ionic strength. We have observed that enzyme activity is adversely affected by high ionic strength buffers such as those recommended for deglycosylation of various glycoproteins and recommend the use of low ionic strength buffers for routine use.

Characterization of the pyoverdines of Azotobacter vinelandii ATCC 12837 with regard to heterogeneity.

Azotobacter vinelandii strain ATCC 12837 produces peptide siderophores of the general class known as pyoverdines. In the past, it was assumed that a single well-defined pyoverdine was produced by each parent microorganism. However, there are a number of reports of incompletely characterized pyoverdines that demonstrate heterogeneity in pyoverdine preparations obtained from a single organism, but the nature of this phenomena has not been explained. This study shows that A. vinelandii does indeed produce more than one pyoverdine and that these compounds differ in their peptide components. The metabolism of these siderophores suggests that only one of them is a true siderophore while the others are metabolic byproducts. It was demonstrated that this phenomenon is likely due to intrinsic limitations of the synthetase complex involved in the biosynthesis of these compounds. Characterization of two of the major pyoverdines produced demonstrated that they are novel compounds, although they belonged to the Azotobacter-type family of pyoverdines.

Stimulation of carbohydrate metabolising enzymes by synthetic hypertrehalosemic peptides in thoracic musculature of the American cockroach, Periplaneta americana.

The ability of the synthetic hypertrehalosemic peptides, HT-I and HT-II, to influence the activities of glycogen phosphorylase, trehalase and hexokinase via elevation of Ca++ and cAMP levels was examined in thoracic musculature of the American cockroach, Periplaneta americana. The peptides effect dose- and time-dependent activation of phosphorylase, trehalase and hexokinase activities that occur concomitantly with elevated levels of intracellular calcium. In addition, HT-I increases the accumulation of cyclic AMP in muscle cells.

Precursor activation in a pyoverdine biosynthesis.

The siderophore produced by Azotobacter vinelandii strain UW belongs to a large family of peptidic siderophores collectively called pyoverdines. The biosynthesis of the peptidyl moiety of this siderophore was shown to involve activation of the constituent amino acids as their adenylates, as demonstrated by amino acid-dependent ATP-[32P]pyrophosphate exchange. The enzyme system responsible for this activation was partially purified by chromatographic techniques.

Studies on lysine:N6-hydroxylation by cell-free systems of Aerobacter aerogenes 62-1.

Electron microscopic examination has revealed the vesicular nature of the membrane component, of the cell-free system of Aerobacter aerogenes 62-1, which catalyses lysine: N6-hydroxylation. Regardless of the orientation of the vesicles, N-hydroxylation process is still stimulated by pyruvate. Both pyruvate oxidation and lysine: N6-hydroxylation were inhibited by protonophores and Gramicidin S.

Peptide: N-glycosidase F: studies on the glycoprotein aminoglycan amidase from Flavobacterium meningosepticum.

Peptide: N-glycosidase from Flavobacterium meningosepticum was isolated in a homogeneous state and its physico-chemical characterization was accomplished. The reliability of the previously recorded assay procedures was assessed. Using an octaglycopeptide derived from ovomucoid a rapid and sensitive FPLC method was developed for the assay of enzymatic activity. Peptide: N-glycosidase was found to effect deglycosylation of glycoproteins bearing complex and/or multiantennary glycans even in their native state. In contrast, glycoproteins with high mannose and/or hybrid carbohydrates required denaturation to become susceptible to deglycosylation by the enzyme.

Direct evidence for the participation of pyruvate in N-hydroxylation of lysine.

The contribution of pyruvate to the formation of N6-acetyl-N6-hydroxylysine by a cell-free system of Aerobacter aerogenes 62-1 involved in the production of the dihydroxamate siderophore, aerobactin, has been assessed by a study of the influence of its analogs as well as of inhibitors of thiamine pyrophosphate-dependent decarboxylation reactions. These studies have provided unequivocal evidence for pyruvate functioning not only as a source of reducing equivalents in the initial step of N-hydroxylation of lysine but also as a precursor of the acetyl moiety in the subsequent conversion of the N-hydroxy amino to its N6-acetyl derivative.

Influence of octopamine on trehalase activity in muscle and hemolymph of the American cockroach, Periplaneta americana L.

Injection of adult male cockroaches (Periplaneta americana) with 10 microliter 1 microM octopamine causes elevated activity of trehalase (alpha, alpha-trehalose glucohydrolase; EC 3.2.1.28) in hemolymph and muscle but not in gut. Tyramine, dopamine and glutamate, at the same concentration, failed to elicit any effect on trehalase activity. Determination of some kinetic parameters for muscle and hemolymph trehalases reveal that octopamine causes an increase in Vmax without any significant alteration in the Km of the enzyme for trehalose. The results are discussed in terms of the physiological significance of octopamine-mediated activation of tissue trehalases.

Stimulation by glutamine of the formation of N6-hydroxylysine in a cell-free extract from Aerobacter aerogenes 62-1.

Glutamine may serve as an activator and/or regulator of the N6-hydroxylase (E.C. 1.14.99) of Aerobacter aerogenes 62-1. Activation and stabilization of N6-hydroxylase activity was observed both in vivo and in vitro. Growth in a glutamine-supplemented medium resulted in (1) maximum N6-hydroxylase activity at an earlier stage of growth and (2) higher N6-hydroxylase activity and continued aerobactin synthesis into stationary phase. Storage of P2 in the presence of L-glutamine (1 mM) significantly increased the lifetime of the labile N6-hydroxylase activity. Inclusion of L-glutamine in the incubation mixture typically resulted in a 2-3-fold activation of the hydroxylase activity. The stimulatory effect of glutamine was independent of and additive to the enhancement of N6-hydroxylation by the active component(s) in the supernatant, S2 fraction. Glutamic acid-gamma-semihydrazide activated slightly in the absence of glutamine but activation of the system by glutamine was decreased by this compound. Azaserine was shown to be an uncompetitive inhibitor with respect to lysine and this inhibition was not reversed by glutamine.