Harry S. Rollema

Protein engineering of lantibiotics

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases be a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibiotics were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.

Biosynthesis and secretion of a precursor of nisin Z by Lactococcus lactis, directed by the leader peptide of the homologous lantibiotic subtilin from Bacillus subtilis

The DNA sequence encoding the leader peptide of the lantibiotic subtilin from Bacillus subtilis was fused to the sequence encoding pronisin Z, and this hybrid gene was expressed in a Lactococcus lactis strain that produces nisin A. This strain simultaneously secreted nisin A and a protein of approximately 6 kDa. Amino acid sequencing of the purified 6 kDa protein and structural analysis of its main tryptic fragment by two‐dimensional 1H‐NMR showed that it consists of the unmodified leader peptide of subtilin, without the N‐terminal methionine residue, linked to a fully matured nisin Z part. The hybrid protein and its main tryptic fragment [ITPQ]‐nisin Z, showed at least 200‐fold lower antimicrobial activities than nisin Z against three different indicator strains.

Reversed-phase high-performance liquid chromatographic separation of bovine κ-casein macropeptide and characterization of isolated fractions

From complex mixtures of non-glycosylated and differently glycosylated caseinomacropeptides (CMP; kappa-casein fragment 106-169; M(r) approximately 7000) various fractions were isolated and further purified by reversed-phase HPLC. The fractions were characterized by mass determination and composition analysis and also used in gel-permeation chromatography and NMR studies to investigate their molecular size behaviour as a function of pH, ionic strength, peptide concentration and degree of glycosylation. No evidence was found for association of any CMP fraction as a function of the experimental conditions applied, which is in contrast with suggestions made in the literature. The increased molecular size (apparent molecular mass approx. 30-45 kDa) is rather explained by a large voluminosity of the molecular species due to internal electrostatic and steric repulsion. Furthermore, the susceptibility of some non-glycosylated and glycosylated CMP fractions to enzymic attack by the Glu-specific endopeptidase from Staphylococcus aureus V8 was studied. Initial rates of proteolysis by this enzyme were independent of the degree of glycosylation. Only in the case of highly glycosylated CMP was further hydrolysis to smaller fragments inhibited. Hydrolytic products were identified by electrospray ionization and fast-atom bombardment mass spectrometry.

The structure of the lantibiotic lacticin 481 produced by Lactococcus lactis : location of the thioether bridges

The lantibiotic lacticin 481 is a bacteriocin produced by Lactococcus lactis ssp. lactis. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether‐bridging lanthionine and 3‐methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A‐FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.

The interaction of chloride ions with human hemoglobin

Abstract Studying the effect of KCl on the Bohr effect of human hemoglobin, it appeared that at low Cl− concentration the alkaline Bohr effect is considerably smaller than it is at a Cl− ion concentration near 0.1 M. The data show that at least part of the Bohr effect, that thus far could not be attributed to a particular residue in hemoglobin, is due to interaction of hemoglobin with anions. The effect of KCl on the Bohr effect shows a striking similarity with the effect of 2,3-diphosphoglycerate (DPG) on the Bohr effect. Based on this a mechanism is proposed which satisfactorily explains the observed salt effect.

Structure and Biological Activity of Chemically Modified Nisin A Species

Nisin, a 34-residue peptide bacteriocin, contains the less common amino acids lanthionine, beta-methyl-lanthionine, dehydroalanine (Dha), and dehydrobutyrine (Dhb). Several chemically modified nisin A species were purified by reverse-phase HPLC and characterized by two-dimensional NMR and electrospray mass spectrometry. Five constituents, [2-hydroxy-Ala5]nisin, [Ile4-amide,pyruvyl-Leu6]des-Dha5-nisin, [Met(O)21]nisin, [Ser33]nisin, and nisin-(1-32)-peptide amide, were found in a commercial nisin sample. A further species, [2-hydroxy-Ala5]nisin-(1-32)-peptide amide, was obtained by freeze drying an acidic nisin solution. These compounds are formed by chemical modification of nisin: the addition of a water molecule to the dehydroalanine residues, which can lead to the cleavage of the polypeptide chain, or the oxidation of methionine residues. The 2-hydroxyalanine-containing products have a limited stability; they are spontaneously converted into the corresponding des-dehydroalanine derivatives. The growth-inhibiting activity of the modified nisins towards different bacteria was determined. The 2-hydroxyalanine-containing species and the des-dehydroalanine derivative show a strong reduction in biological activity as compared to native nisin. [Met(O)21]nisin and [Ser33]nisin show moderate or no reduction in biological activity.

NMR and circular dichroism studies of the lantibiotic nisin in non-aqueous environments

The lantibiotic, nisin, which is known to interact with membranes of certain Gram‐positive bacteria, was studied in three model systems which mimic a membrane‐like environment, i.e. a mixture of trifluoroethanol and water, or micelles of sodium dodecyl sulfate or dodecylphosphocholine. The 1H NMR spectra of nisin in the non‐aqueous environments, at 40°C and pH 3.5, have been assigned completely. The CD and NMR results indicate that the conformation of nisin in the three non‐aqueous environments differs from that in aqueous solution, and that the conformation in the two micellar systems is similar. The major conformational changes, relative to nisin in aqueous solution, occur in the N‐terminus.

Protein Engineering and Biosynthesis of Nisin and Regulation of Transcription of the Structural nisA Gene

Abstract The lantibiotic nisin, produced by Lactococcus lactis , is an antimicrobial peptide characterized by the presence of three unsaturated amino acid side chains (two dehydroalanines and one dehydrobutyrine) and five (β-methyl)lanthionine rings, which are formed post-translationally. Nisin is widely used in the food industry as a preservative, since it inhibits the growth of unwanted gram-positive bacteria. One of the objectives of our research is to get insight in the complex biosynthesis and regulation of production of nisin. The structure and function of several biosynthetic genes were studied by making gene disruptions and by subsequently investigating their effects on nisin gene regulation, biosynthesis, secretion and immunity. An exciting finding is that nisin itself, when added to the culture medium, can induce the transcription of its own structural gene. Another goal is to design and produce altered nisin molecules with desirable properties by protein engineering. In addition to previously reported mutant nisins with improved stability, solubility or activity, recent results on the protein engineering of residues Ile1, Dhb2, AlaS3, Lys12, AbuS13, Met17, Asn20 and Met21 indicate that (i) residue 1 can be replaced without dramatic loss of activity; (ii) the presence of a Thr residue at position 2 significantly lowers the antimicrobial potency, whereas the presence of a Dha residue at position 2 improves activity; (iii) the replacement ofAlaS3 by AbuS leads to a dramatic loss of activity, probably due to a conformational change in the first lanthionine ring; (iv) the integrity and hydrophobicity of ring 3 are important for antimicrobial activity; and (v) the hinge region between rings 3 and 4 is important but not essential for antimicrobial activity.

The kinetics of carbon monoxide binding to partially reduced methemoglobin

Abstract The pulse radiolysis technique has been used to study the kinetics of the CO binding to partially reduced methemoglobin. Experiments with horse heart metmyoglobin show that this technique gives results which are in good agreement with those obtained by other methods. The kinetics of the CO binding to partially reduced methemoglobin show two phases, whose amplitudes appear to depend on the degree of reduction in such a way that they can be attributed to methemoglobin molecules with one or two reduced heme groups. In the presence of inositol hexaphosphate the rate of CO binding to partially reduced methemoglobin decreases strongly. With inositol hexaphosphate a slight biphasic behavior is observed independent of the degree of reduction.

Pulse-radiolytic studies on the spin-state transitions in aquomethemoglobin after reduction of a single heme group

We have previously observed a transient state (halftime ‘L 15 ps) in aquomethemoglobin with an absorption maximum near 420 nm after rapid reduction of a single ferric heme group by hydrated electrons [l] . This observation has been confirmed [2,3] . Moreover, this microsecond process disappeared in the presence of Ins-P6 [l] . On binding this organic phosphate, metHb changes its quaternary conformation from the Rto the T-state [4]. Therefore, we concluded that in the absence of an allosteric effector the reduction reaction of one heme group in metHb by hydrated electrons was followed by a quaternary conformational change (R + T transition). spin-state. The subsequent transition of the ferrous low spin to the stable ferrous high spin-state proceeds with a half-time that depends on the solvent condition. On the basis of these results we have postulated a two-step reduction mechanism for aquomethemoglobin.