Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633.

Subtilin is a ribosomally synthesized peptide antibiotic produced by Bacillus subtilis ATCC 6633. Recently, we reported regarding genes spaB, spaT, and spaC (C. Klein, C. Kaletta, N. Schnell, and K.-D. Entian, Appl. Environ. Microbiol. 58:132-142, 1992) which are involved in the biosynthesis of subtilin, and genes spaR and spaK (C. Klein, C. Kaletta, and K.-D. Entian, Appl. Environ. Microbiol. 59:296-303, 1993), which regulate subtilin biosynthesis via a histidine kinase/response regulator system. Further sequence analysis revealed the presence of three additional open reading frames, spaI, spaF, and spaG, downstream of the structural gene spaS. The spaI gene encodes a hydrophilic 19.3-kDa lipoprotein containing a consensus signal sequence, indicating that this protein might be membrane anchored. A similar gene, nisI, has been identified in the nisin producer. SpaF shows strong homology to members of the family of ABC transporters. spaG encodes a hydrophobic protein which might form the active transporter together with SpaF. Gene disruption mutants in all three genes were still able to produce subtilin; however, these mutants were more sensitive to subtilin than the wild-type strain. These results show that these genes are involved in the immunity mechanism of the producer strain. A similar involvement of an ABC transporter in the self-protection mechanism has been described for the McbE and McbF transporter, which confers immunity against microcin B17 in Escherichia coli. Mutants containing mutations in the genes spaR and spaK, which are responsible for regulation of subtilin biosynthesis, also became more sensitive to subtilin.(ABSTRACT TRUNCATED AT 250 WORDS)     

The action of the bacterial toxin, microcin B17, on DNA gyrase

Microcin B17 (MccB17) is a peptide-based bacterial toxin that targets DNA gyrase, the bacterial enzyme that introduces supercoils into DNA. The site and mode of action of MccB17 on gyrase are unclear. We review what is currently known about MccB17-gyrase interactions and summarise approaches to understanding its mode of action that involve modification of the toxin. We describe experiments in which treatment of the toxin at high pH leads to the deamidation of two asparagine residues to aspartates. The modified toxin was found to be inactive in vivo and in vitro, suggesting that the Asn residues are essential for activity. Following on from these studies we have used molecular modelling to suggest a 3D structure for microcin B17. We discuss the implications of this model for MccB17 action and investigate the possibility that it binds metal ions.     

The highly conserved TldD and TldE proteins of Escherichia coli are involved in microcin B17 processing and in CcdA degradation

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains carrying the pMccB17 plasmid. MccB17 is synthesized as a precursor containing an amino-terminal leader peptide that is cleaved during maturation. Maturation requires the product of the chromosomal tldE (pmbA) gene. Mature microcin is exported across the cytoplasmic membrane by a dedicated ABC transporter. In sensitive cells, MccB17 targets the essential topoisomerase II DNA gyrase. Independently, tldE as well as tldD mutants were isolated as being resistant to CcdB, another natural poison of gyrase encoded by the ccd poison-antidote system of plasmid F. This led to the idea that TldD and TldE could regulate gyrase function. We present in vivo evidence supporting the hypothesis that TldD and TldE have proteolytic activity. We show that in bacterial mutants devoid of either TldD or TldE activity, the MccB17 precursor accumulates and is not exported. Similarly, in the ccd system, we found that TldD and TldE are involved in CcdA and CcdA41 antidote degradation rather than being involved in the CcdB resistance mechanism. Interestingly, sequence database comparisons revealed that these two proteins have homologues in eubacteria and archaebacteria, suggesting a broader physiological role.     

Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17

In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.     

The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase.

Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT----GC transition at position 2251 of the gene, which produces a Trp751----Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell-free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double-strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well-defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17-resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme-DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.     

Evidence for the role of DNA strand passage in the mechanism of action of microcin B17 on DNA gyrase

Microcin B17 (MccB17) is a DNA gyrase poison; in previous work, this bacterial toxin was found to slowly and incompletely inhibit the reactions of supercoiling and relaxation of DNA by gyrase and to stabilize the cleavage complex, depending on the presence of ATP and the DNA topology. We now show that the action of MccB17 on the gyrase ATPase reaction and cleavage complex formation requires a linear DNA fragment of more than 150 base pairs. MccB17 is unable to stimulate the ATPase reaction by stabilizing the weak interactions between short linear DNA fragments (70 base pairs or less) and gyrase, in contrast with the quinolone ciprofloxacin. However, MccB17 can affect the ATP-dependent relaxation of DNA by gyrase lacking its DNA-wrapping or ATPase domains. From these findings, we propose a mode of action of MccB17 requiring a DNA molecule long enough to allow the transport of a segment through the DNA gate of the enzyme. Furthermore, we suggest that MccB17 may trap a transient intermediate state of the gyrase reaction present only during DNA strand passage and enzyme turnover. The proteolytic signature of MccB17 from trypsin treatment of the full enzyme requires DNA and ATP and shows a protection of the C-terminal 47-kDa domain of gyrase, indicating the involvement of this domain in the toxin mode of action and consistent with its proposed role in the mechanism of DNA strand passage. We suggest that the binding site of MccB17 is in the C-terminal domain of GyrB.     

The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents

Microcin B17 (MccB17) is an antibacterial peptide produced by strains of Escherichia coli harboring the plasmid-borne mccB17 operon. MccB17 possesses many notable features. It is able to stabilize the transient DNA gyrase–DNA cleavage complex, a very efficient mode of action shared with the highly successful fluoroquinolone drugs. MccB17 stabilizes this complex by a distinct mechanism making it potentially valuable in the fight against bacterial antibiotic resistance. MccB17 was the first compound discovered from the thiazole/oxazole-modified microcins family and the linear azole-containing peptides; these ribosomal peptides are post-translationally modified to convert serine and cysteine residues into oxazole and thiazole rings. These chemical moieties are found in many other bioactive compounds like the vitamin thiamine, the anti-cancer drug bleomycin, the antibacterial sulfathiazole and the antiviral nitazoxanide. Therefore, the biosynthetic machinery that produces these azole rings is noteworthy as a general method to create bioactive compounds. Our knowledge of MccB17 now extends to many aspects of antibacterial–bacteria interactions: production, transport, interaction with its target, and resistance mechanisms; this knowledge has wide potential applicability. After a long time with limited progress on MccB17, recent publications have addressed critical aspects of MccB17 biosynthesis as well as an explosion in the discovery of new related compounds in the thiazole/oxazole-modified microcins/linear azole-containing peptides family. It is therefore timely to summarize the evidence gathered over more than 40 years about this still enigmatic molecule and place it in the wider context of antibacterials.     

The Ile13 residue of microcin J25 is essential for recognition by the receptor FhuA, but not by the inner membrane transporter SbmA

Entry of the peptide antibiotic microcin J25 (MccJ25) into target cells is mediated by the outer membrane receptor FhuA and the inner membrane protein SbmA. The latter also transports MccB17 into the cell cytoplasm. Comparison of MccJ25 and MccB17 revealed a tetrapeptide sequence (VGIG) common to both antibiotics. We speculated that this structural feature in MccJ25 could be a motif recognized by SbmA. To test this hypothesis, we used a MccJ25 variant in which the isoleucine in VGIG (position 13 in the MccJ25 sequence) was replaced by lysine (I13K). In experiments in which the FhuA receptor was bypassed, the substituted microcin showed an inhibitory activity similar to that of the wild-type peptide. Moreover, MccJ25 interfered with colicin M uptake by FhuA in a competition assay, while the I13K mutant did not. From these results, we propose that the Ile¹³ residue is only required for interaction with FhuA, and that VGIG is not a major recognition element by SbmA.     

Influence of aeration and carbon source on production of microcin B17 by Escherichia coli ZK650

Previous studies [Connell et al. (1987) Mol Microbiol 1: 195–201] have shown that expression of the microcin B17 (MccB17) promoter is inversely related to the growth rate of the culture, when slower growth was brought about by limitation of sources of carbon, nitrogen or phosphorus. When we used oxygen limitation to decrease growth in a glucose-based chemically defined medium, we found specific MccB17 production to be positively related to growth rate and extent. On the other hand, when we examined various nutritional variations of media, specific production of MccB17 showed a negative relationship to growth rate and extent, as would be predicted by the findings of Connell et al. (1987). Glucose, glycerol and acetate were found to repress MccB17 production; succinate was not repressive. Succinate is an excellent carbon source for production of MccB17 since high levels can be used with no or little interference in product synthesis. Received: 26 November 1996 / Accepted: 7 December 1996     

The action of the bacterial toxin microcin B17. Insight into the cleavage-religation reaction of DNA gyrase

We have examined the effects of the bacterial toxin microcin B17 (MccB17) on the reactions of Escherichia coli DNA gyrase. MccB17 slows down but does not completely inhibit the DNA supercoiling and relaxation reactions of gyrase. A kinetic analysis of the cleavage-religation equilibrium of gyrase was performed to determine the effect of the toxin on the forward (cleavage) and reverse (religation) reactions. A simple mechanism of two consecutive reversible reactions with a nicked DNA intermediate was used to simulate the kinetics of cleavage and religation. The action of MccB17 on the kinetics of cleavage and religation was compared with that of the quinolones ciprofloxacin and oxolinic acid. With relaxed DNA as substrate, only a small amount of gyrase cleavage complex is observed with MccB17 in the absence of ATP, whereas the presence of the nucleotide significantly enhances the effect of the toxin on both the cleavage and religation reactions. In contrast, ciprofloxacin, oxolinic acid, and Ca2+ show lesser dependence on ATP to stabilize the cleavage complex. MccB17 enhances the overall rate of DNA cleavage by increasing the forward rate constant (k2) of the second equilibrium. In contrast, ciprofloxacin increases the amount of cleaved DNA by a combined effect on the forward and reverse rate constants of both equilibria. Based on these results and on the observations that MccB17 only slowly inhibits the supercoiling and relaxation reactions, we suggest a model of the interaction of MccB17 with gyrase.     

Fragments of the Bacterial Toxin Microcin B17 as Gyrase Poisons

Fluoroquinolones are very important drugs in the clinical antibacterial arsenal; their success is principally due to their mode of action: the stabilisation of a gyrase-DNA intermediate (the cleavage complex), which triggers a chain of events leading to cell death. Microcin B17 (MccB17) is a modified peptide bacterial toxin that acts by a similar mode of action, but is unfortunately unsuitable as a therapeutic drug. However, its structure and mechanism could inspire the design of new antibacterial compounds that are needed to circumvent the rise in bacterial resistance to current antibiotics. Here we describe the investigation of the structural features responsible for MccB17 activity and the identification of fragments of the toxin that retain the ability to stabilise the cleavage complex.     

The maturation pathway of microcin B17, a peptide inhibitor of DNA gyrase

The maturation pathway of microcin B17 (MccB17), a ribosomally synthesized peptide antibiotic which inhibits DNA gyrase, has been characterized. Synthesis of MccB17 involves several steps beginning with the translation of the MccB17 structural gene, mcbA, to yield a 69 amino acid precursor, preMccB17. PreMccB17 is then modified and folded by the action of three gene products, McbBCD, to yield proMccB17. Mutations in mcbA were isolated that permit modifications of the resulting mutant peptides, but prevent folding, suggesting that modification and folding are sequential steps. ProMccB17 is subsequently converted to MccB17 by removal of the W-terminal 26-amino-acid leader by a chromosomally encoded protease. Removal of the leader resulted in aggregation of the peptide, suggesting that the leader may function to maintain peptide solubility during synthesis in the cell. Finally, polyclonal antibodies raised against MccB17 recognize both MccB17 and proMccB17, but do not recognize preMccB17. This demonstrates the dramatic structural changes that result from the modifications and has been used to distinguish intermediates in the steps of maturation.     

sbmC, a stationary-phase induced SOS Escherichia coli gene, whose product protects cells from the DNA replication inhibitor microcin B17

Microcin B17 (MccB17) is a ribosomally synthesized peptide antibiotic of 43 amino acids that induces double-strand breaking of DNA in a DNA gyrase-dependent reaction. As a consequence, the SOS regulon is induced and massive DNA degradation occurs. In this work we have characterized an Escherichia coli gene, sbmC , that in high copy number determines high cell resistance to MccB17. sbmC encodes a cytoplasmic polypeptide of 157 amino acids ( M_r , 18 095) that has been visualized in SDS—polyacrylamide gels. The gene is located at min 44 of the E. coli genetic map, close to the sbcB gene. sbmC expression is induced by DNA-damaging agents and, also, by the entry of cells into the stationary growth phase. A G → T transversion at the fifth nucleotide of the quasicanonical LexA-box preceding the gene makes recA cells 16-fold more resistant to exogenous MccB17. The gene product, SbmC, also blocks MccB17 export from producing cells. Altogether, our results suggest that SbmC recognizes and sequesters MccB17 in a reversible way.     

The leader peptide is essential for the post-translational modification of the DNA-gyrase inhibitor microcin B17

Microcin B17 (MccB17) is a ribosomally encoded DNA-gyrase inhibitor. Ribosomally encoded antibiotics are derived from precursors containing an N-terminal leader, which is removed during maturation, and a C-terminal structural peptide. PreMccB17, the translational product of mcbA , is modified into proMccB17 by the action of three enzymes, McbB, McbC, and McbD. A chromosomally encoded peptidase then converts proMccB17 into MccB17. The role of McbB, McbC, and McbD is to convert glycine, cysteine, and serine residues present in preMccB17 into four thiazole and four oxazole rings. Using a modification-specific antibody rather than antimicrobial activity, we show that the 26-amino-acid N-terminal leader of preMccB17 is essential for the conversion of preMccB17 into proMccB17. Neither a preMccB17 peptide lacking the leader nor a preMccB17–β-galactosidase fusion lacking the leader are post-translationally modified.     

Molecular characterization of pmbA, an Escherichia coli chromosomal gene required for the production of the antibiotic peptide MccB17

Microcin B17 (MccB17) is a peptide antibiotic produced by Escherichia coli strains harbouring plasmid pMccB17. We have isolated two mutations that strongly reduce the production of MccB17. These mutations, which map at 96 min on the E. coli chromosome, define a new gene that we have called pmbA. A chromosomal DNA fragment of about 13 kb, including the wild-type pmbA allele, was cloned into a mini-Mu plasmid vector. pmbA was located within the cloned DNA fragment by insertional mutagenesis and deletion analysis. The nucleotide sequence of a 1.7 kb DNA region containing the gene was determined. pmbA encodes a hydrophilic protein of 450-amino-acid residues with a predicted molecular size of 48375D, which was visualized in polyacrylamide gels. Protein profiles of cellular envelope and soluble fractions from cells with plasmids overproducing PmbA indicated that it is cytoplasmic. Physiological experiments suggested that pmbA mutants synthesize a molecule (pro-MccB17) able to inhibit DNA replication but unable to be released from cells. We propose that PmbA facilitates the secretion of the antibiotic by completing its maturation.     

Identification of functional toxin/immunity genes linked to contact-dependent growth inhibition (CDI) and rearrangement hotspot (Rhs) systems

Bacterial contact-dependent growth inhibition (CDI) is mediated by the CdiA/CdiB family of two-partner secretion proteins. Each CdiA protein exhibits a distinct growth inhibition activity, which resides in the polymorphic C-terminal region (CdiA-CT). CDI(+) cells also express unique CdiI immunity proteins that specifically block the activity of cognate CdiA-CT, thereby protecting the cell from autoinhibition. Here we show that many CDI systems contain multiple cdiA gene fragments that encode CdiA-CT sequences. These "orphan" cdiA-CT genes are almost always associated with downstream cdiI genes to form cdiA-CT/cdiI modules. Comparative genome analyses suggest that cdiA-CT/cdiI modules are mobile and exchanged between the CDI systems of different bacteria. In many instances, orphan cdiA-CT/cdiI modules are fused to full-length cdiA genes in other bacterial species. Examination of cdiA-CT/cdiI modules from Escherichia coli EC93, E. coli EC869, and Dickeya dadantii 3937 confirmed that these genes encode functional toxin/immunity pairs. Moreover, the orphan module from EC93 was functional in cell-mediated CDI when fused to the N-terminal portion of the EC93 CdiA protein. Bioinformatic analyses revealed that the genetic organization of CDI systems shares features with rhs (rearrangement hotspot) loci. Rhs proteins also contain polymorphic C-terminal regions (Rhs-CTs), some of which share significant sequence identity with CdiA-CTs. All rhs genes are followed by small ORFs representing possible rhsI immunity genes, and several Rhs systems encode orphan rhs-CT/rhsI modules. Analysis of rhs-CT/rhsI modules from D. dadantii 3937 demonstrated that Rhs-CTs have growth inhibitory activity, which is specifically blocked by cognate RhsI immunity proteins. Together, these results suggest that Rhs plays a role in intercellular competition and that orphan gene modules expand the diversity of toxic activities deployed by both CDI and Rhs systems.     

Computational analysis of the first biheterocyclization site of the antibiotic microcin B17

Microcin B 17 (MccB17) undergoes an enzyme catalyzed posttranslational modification to form four oxazole and four thiazole rings. Four of these rings form 4,2 - connected biheterocyclic functionalities. In this study, the hexapeptide sequence surrounding the first biheterocyclization site of microcin B17 was examined using computational calculations and database analysis to see if it was preorganized for cyclization in a manner similar to that found in the autocatalytic posttranslational cyclization of Green Fluorescent Protein (GFP). Attention was focused on the intermolecular distances between the sulfur and oxygen atoms of the cysteine and serine residues and the carbonyl carbons which they attack in the ring formation. Conformational searches located some low energy conformations that contained relatively short oxygen to carbonyl carbon distances, which indicated that the oxazole forming fragment in microcin B17 is preorganized for cyclization. However, the lack of any clear patterns for the sulfur to carbon distances show that the side-chain of cysteine does not adopt any low energy conformations that are geometrically preorganized for cyclization. The MccB17 synthetase enzyme complex which catalyzes the cyclization process therefore has both steric and electronic functions. The data obtained in this investigation is in agreement with empirical data which shows that biheterocyclization will only occur if the thiazole forms before the oxazole.     

How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic

The 39‐kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a ‘Trojan horse’ antibiotic. The approximately 260‐residue C‐terminal region of MccB is homologous to ubiquitin‐like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB‐catalysed C‐terminal MccA‐acyl‐adenylation is reminiscent of the E1‐catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C‐terminal di‐glycine sequence, MccB's substrate, MccA, is a short peptide with an essential C‐terminal Asn. Furthermore, after an intramolecular rearrangement of MccA‐acyl‐adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate‐linked analogue of acyl‐adenylated aspartic acid. We report six‐crystal structures of MccB in apo, substrate‐, intermediate‐, and inhibitor‐bound forms. Structural and kinetic analyses reveal a novel‐peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic‐active site for catalysing dual adenosine triphosphate‐consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two‐step activation for generating the peptidyl‐antibiotic MccC7.     

Structure and organization of plasmid genes required to produce the translation inhibitor microcin C7.

The translation inhibitor microcin C7 (MccC7) is a linear heptapeptide whose N terminus has been replaced by an N-formyl group and whose C terminus has been replaced by the phosphodiester of 5'-adenylic acid and n-aminopropanol (J. I. Guijarro, J. E. González-Pastor, F. Baleux, J. L. San Millán, M. A. Castilla, M. Rico, F. Moreno, and M. Delepierre, J. Biol. Chem. 270:23520-23532, 1995). MccC7 production and immunity determinants lie on a 6.2-kb region of the Escherichia coli plasmid pMccC7. This region was entirely sequenced. It contains six open reading frames, which were shown to be true genes by different complementary approaches. Five genes, mccABCDE, which are transcribed in the same direction, are required to produce mature extracellular microcin. The sixth gene, mccF, adjacent to mccE, is transcribed in the opposite direction and encodes specific self-immunity. Genes mccA to -E constitute an operon transcribed from a promoter (mccp) located upstream of mccA. mccA is 21 nucleotides long and encodes the unmodified heptapeptide (J. E. González-Pastor, J. L. San Millán, and F. Moreno, Nature [London] 369:281, 1994). A comparison of predicted gene polypeptide products with those included in databases shows that an 81-amino-acid stretch of MccB is strikingly homologous to fragments of the same length of proteins ThiF and ChlN from E. coli, HesA from Anabaena sp. strain PCC7120, and UBA1, the ubiquitin-activating enzyme from different eukaryotic species. MccC displays several hydrophobic domains, suggesting a transmembrane location. The carboxyl end of MccE displays 41.2% identity with RimL, a protein required to acetylate the ribosome protein L12 from E. coli. In the absence of the other mcc genes, mccA impairs the growth of host cells, suggesting that unmodified MccA has antibiotic activity. A model for MccC7 biosynthesis, export, and immunity is proposed.     

Computational Analysis of the First Biheterocyclization Site of the Antibiotic Microcin B17

Abstract

Microcin B17 (MccB17) undergoes an enzyme catalyzed posttranslational modification to form four oxazole and four thiazole rings. Four of these rings form 4,2—connected bihete-rocyclic functionalities. In this study, the hexapeptide sequence surrounding the first bihete- rocyclization site of microcin B17 was examined using computational calculations and database analysis to see if it was preorganized for cyclization in a manner similar to that found in the autocatalytic posttranslational cyclization of Green Fluorescent Protein (GFP). Attention was focused on the intermolecular distances between the sulfur and oxygen atoms of the cysteine and serine residues and the carbonyl carbons which they attack in the ring formation. Conformational searches located some low energy conformations that contained relatively short oxygen to carbonyl carbon distances, which indicated that the oxazole forming fragment in microcin B17 is preorganized for cyclization. However, the lack of any clear patterns for the sulfur to carbon distances show that the side-chain of cysteine does not adopt any low energy conformations that are geometrically preorganized for cyclization. The MccB17 synthetase enzyme complex which catalyzes the cyclization process therefore has both steric and electronic functions. The data obtained in this investigation is in agreement with empirical data which shows that biheterocyclization will only occur if the thiazole forms before the oxazole.