The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains.

The cyclic decapeptide antibiotic tyrocidine is produced by Bacillus brevis ATCC 8185 on an enzyme complex comprising three peptide synthetases, TycA, TycB, and TycC (tyrocidine synthetases 1, 2, and 3), via the nonribosomal pathway. However, previous molecular characterization of the tyrocidine synthetase-encoding operon was restricted to tycA, the gene that encodes the first one-module-bearing peptide synthetase. Here, we report the cloning and sequencing of the entire tyrocidine biosynthesis operon (39.5 kb) containing the tycA, tycB, and tycC genes. As deduced from the sequence data, TycB (404,562 Da) consists of three modules, including an epimerization domain, whereas TycC (723,577 Da) is composed of six modules and harbors a putative thioesterase domain at its C-terminal end. Each module incorporates one amino acid into the peptide product and can be further subdivided into domains responsible for substrate adenylation, thiolation, condensation, and epimerization (optional). We defined, cloned, and expressed in Escherichia coli five internal adenylation domains of TycB and TycC. Soluble His6-tagged proteins, ranging from 536 to 559 amino acids, were affinity purified and found to be active by amino acid-dependent ATP-PPi exchange assay. The detected amino acid specificities of the investigated domains manifested the colinear arrangement of the peptide product with the respective module in the corresponding peptide synthetases and explain the production of the four known naturally occurring tyrocidine variants. The Km values of the investigated adenylation domains for their amino acid substrates were found to be comparable to those published for undissected wild-type enzymes. These findings strongly support the functional integrities of single domains within multifunctional peptide synthetases. Directly downstream of the 3' end of the tycC gene, and probably transcribed in the tyrocidine operon, two tandem ABC transporters, which may be involved in conferring resistance against tyrocidine, and a putative thioesterase were found.     

Mutational analysis of the C-domain in nonribosomal peptide synthesis.

The initial condensation event in the nonribosomal biosynthesis of the peptide antibiotics gramicidin S and tyrocidine A takes place between a phenylalanine activating racemase GrsA/TycA and the first proline-activating module of GrsB/TycB. Recently we established a minimal in vitro model system for NRPS with recombinant His6-tagged GrsA (GrsAPhe-ATE; 127 kDa) and TycB1 (TycB1Pro-CAT; 120 kDa) and demonstrated the catalytic function of the C-domain in TycB1Pro-CAT to form a peptide bond between phenylalanine and proline during diketopiperazine formation (DKP). In this work we took advantage of this system to identify catalytically important residues in the C-domain of TycB1Pro-CAT using site-directed mutagenesis and peptide mapping. Mutations in TycB1Pro-CAT of 10 strictly conserved residues among 80 other C-domains with potential catalytic function, revealed that only R62A, H147R and D151N are impaired in peptide-bond formation. All other mutations led to either unaffected (Q19A, C154A/S, Y166F/W and R284A) or insoluble proteins (H146A, R67A and W202L). Although 100 nm of the serine protease inhibitors N-alpha-tosyl-l-phenylalanylchloromethane or phenylmethanesulfonyl fluoride completely abolished DKP synthesis, no covalently bound inhibitor derivatives in the C-domain could be identified by peptide mapping using HPLC-MS. Though the results do not reveal a particular mechanism for the C-domain, they exhibit a possible way of catalysis analogous to the functionally related enzymes chloramphenicol acetyltransferase and dihydrolipoyl transacetylase. Based on this, we propose a mechanism in which one catalytic residue (H147) and two other structural residues (R62 and D151) are involved in amino-acid condensation.     

Timing of epimerization and condensation reactions in nonribosomal peptide assembly lines: kinetic analysis of phenylalanine activating elongation modules of tyrocidine synthetase B

The cyclic decapeptide antibiotic tyrocidine has D-Phe residues at positions 1 and 4, produced during peptide chain growth from L-Phe residues by 50 kDa epimerase (E) domains embedded, respectively, in the initiation module (TycA) and the TycB3 module of the three-subunit (TycABC), 10-module nonribosomal peptide synthetase. While the initiation module clearly epimerizes the aminoacyl thioester Phe1-S-TycA intermediate, the timing of epimerization versus peptide bond condensation at internal E domains has been less well characterized in nonribosomal peptide synthetases. In this study, we use rapid quench techniques to evaluate a three-domain (ATE) and a four-domain version (CATE) of the TycB3 module and a six-domain fragment (ATCATE) of the TycB2(-3) bimodule to measure the ability of the E domain in the TycB3 module to epimerize the aminoacyl thioester Phe-S-TycB3 and the dipeptidyl-S-enzyme (L-Phe-L-Phe-S-TycB3 if L-Phe-D-Phe-S-TycB3). The chiralities of the Phe-S-enzyme and Phe-Phe-S-enzyme species over time were determined by hydrolysis and chiral TLC separations, allowing for the clear conclusion that epimerization in the internal TycB3 module occurs preferentially on the peptidyl-S-enzyme rather than the aminoacyl-S-enzyme, by a factor of about 3000/1. In turn, this imposes constraints on the chiral selectivity of the condensation (C) domains immediately upstream and downstream of E domains. The stereoselectivity of the upstream C domain was shown to be L-selective at both donor and acceptor sites ((L)C(L)) by site-directed mutagenesis studies of an E domain active site residue and using the small-molecule surrogate D-Phe-Pro-L-Phe-N-acetylcysteamine thioester (D-Phe-Pro-L-Phe-SNAC) and D-Phe-Pro-D-Phe-SNAC as donor probes.     

Control of directionality in nonribosomal peptide synthesis: role of the condensation domain in preventing misinitiation and timing of epimerization

Product assembly by nonribosomal peptide synthetases (NRPS) is initiated by starter modules that comprise an adenylation (A) and a peptidyl carrier protein (PCP) domain. Elongation modules of NRPS have in addition a condensation (C) domain that is located upstream of the A domain. They cannot initiate peptide bond formation. To understand the role of domain arrangements and the influence of the domains present upstream of the A domains of the elongation modules of TycB on the initiation and epimerization activities, we constructed a set of proteins derived from the tyrocidine synthetases of Bacillus brevis, which represent several N-terminal truncations of TycB and the first module of TycC. The latter was fused with the thioesterase domain (Te) to give TycC(1)-CAT-Te and to ensure product turnover. TycB(2)(-)(3)-AT.CATE and TycB(3)-ATE, lacking an N-terminal C domain, were capable of initiating peptide synthesis and epimerizing. In contrast, the corresponding constructs with a cognate N-terminal C domain, TycB(2)(-)(3)-T.CATE and TycB(3)-CATE, were strongly reduced in initiation and epimerization. Evidence is also provided that this reduction is due to substrate binding in an enantioselective binding pocket at the acceptor position of the C domains. By using TycB(2)(-)(3)-AT.CATE and TycB(3)-ATE, we were able to turn an elongation module into an initiation module, and to establish an in-trans system for the formation of new di- and tripeptides with recombinant NRPS modules. We also show that epimerization domains of elongation modules can in principle epimerize both aminoacyl-S-Ppant (TycB(3)-ATE) and peptidyl-S-Ppant (TycB(2)(-)(3)-AT.CATE) substrates, although the efficiency for epimerizing the noncognate aminoacyl-S-Ppant substrates appears to be lowered.     

Recognition of hybrid peptidyl carrier proteins/acyl carrier proteins in nonribosomal peptide synthetase modules by the 4'-phosphopantetheinyl transferases AcpS and Sfp

The acyl carrier proteins (ACPs) of fatty acid synthase and polyketide synthase as well as peptidyl carrier proteins (PCPs) of nonribosomal peptide synthetases are modified by 4'-phosphopantetheinyl transferases from inactive apo-enzymes to their active holo forms by transferring the 4'-phosphopantetheinyl moiety of coenzyme A to a conserved serine residue of the carrier protein. 4'-Phosphopantetheinyl transferases have been classified into two types; the AcpS type accepts ACPs of fatty acid synthase and some ACPs of type II polyketide synthase as substrates, whereas the Sfp type exhibits an extraordinarily broad substrate specificity. Based on the previously published co-crystal structure of Bacillus subtilis AcpS and ACP that provided detailed information about the interacting residues of the two proteins, we designed a novel hybrid PCP by replacing the Bacillus brevis TycC3-PCP helix 2 with the corresponding helix of B. subtilis ACP that contains the interacting residues. This was performed for the PCP domain as a single protein as well as for the TycA-PCP domain within the nonribosomal peptide synthetase module TycA from B. brevis. Both resulting proteins, designated hybrid PCP (hPCP) and hybrid TycA (hTycA), were modified in vivo during heterologous expression in Escherichia coli (hPCP, 51%; hTycA, 75%) and in vitro with AcpS as well as Sfp to 100%. The designated hTycA module contains two other domains: an adenylation domain (activating phenylalanine to Phe-AMP and afterward transferring the Phe to the PCP domain) and an epimerization domain (converting the PCP-bound l-Phe to d-Phe). We show here that the modified PCP domain of hTycA communicates with the adenylation domain and that the co-factor of holo-hPCP is loaded with Phe. However, communication between the hybrid PCP and the epimerization domain seems to be disabled. Nevertheless, hTycA is recognized by the next proline-activating elongation module TycB1 in vitro, and the dipeptide is formed and released as diketopiperazine.     

Chirality of peptide bond-forming condensation domains in nonribosomal peptide synthetases: the C5 domain of tyrocidine synthetase is a (D)C(L) catalyst

Nonribosomal peptides (NRP) such as the antibiotic tyrocidine have D-amino acids, introduced by epimerase (E) domains embedded within modules of the enzymatic assembly lines. We predict that the peptide bond-forming condensation (C) domains immediately downstream of E domains are D-specific for the peptidyl donor and L-specific for the aminoacyl acceptor ((D)C(L)). To validate this prediction and establish that the C(5) domain of tyrocidine synthetase is indeed (D)C(L), the apoT (thiolation) forms of module 4 (TycB(3) AT(4)E) and module 5 (TycC(1) C(5)AT(5)) were expressed. T(5) was posttranslationally primed with CoASH to introduce the HS-pantetheinyl group and autoaminoacylated with radiolabeled L-Asn* or L-Asp*. Alternate donor substrates were introduced by priming apo AT(4)E with synthetically prepared tetrapeptidyl-CoA's differing in the chirality of Phe-4, D-Phe-L-Pro-L-Phe-L-Phe-CoA, and D-Phe-L-Pro-L-Phe-D-Phe-CoA. The tetrapeptidyl-S-T(4) and L-Asp-S-T(5) were studied for peptide bond formation and chain translocation by C(5) to yield pentapeptidyl-S-T(5), whose chirality (D-L-L-D-L- vs D-L-L-L-L-) was assayed by thioester cleavage and chiral chromatography of the released pentapeptides. Only the D-Phe-4 pentapeptidyl-S-T(5) was generated, implying that only D-L-L-D-S-T(4) was utilized, proving C(5) is indeed a (D)C(L) catalyst. Furthermore, a mutant with an inactive E domain transferred tetrapeptide only when loaded with D-Phe-4 tetrapeptidyl donor, not L-Phe-4, confirming that in the wild-type assembly line C(5) only transfers D-L-L-L-tetrapeptidyl-S-T(4) after in situ epimerization by the E domain. These results contrast the observation that C(5) can make both L-Phe-L-Asn and D-Phe-L-Asn when assayed with Phe as the donor substrate. Hence, utilizing an aminoacyl-S-T(4) versus the natural peptidyl-S-T(4) donor produced misleading information regarding the specificity of the condensation domain.     

Aminoacyl-coenzyme A synthesis catalyzed by adenylation domains

Adenylate forming enzymes play an important role in nature as they are involved in a number of essential biochemical pathways. In this study, we investigated the ability of a set of structurally related recombinant bacterial adenylate forming enzymes derived from nonribosomal peptide synthetases for their ability to synthesize acyl-CoAs in vitro. Adenylation-domains normally transfer their reactive aminoacyl-adenylates onto the covalently attached 4'-phosphopantetheine moiety of small carrier proteins. In detail, DltA, DhbE, GrsA-A, TycB(3)-A, and TycC(3)-A were investigated for their ability to synthesize acyl-CoAs. As reference, acetyl-CoA-synthetase (Acs) of B. subtilis was utilized, which naturally synthesizes acetyl-CoA from acetate, CoA-SH and ATP. Interestingly, all enzymes were capable of producing acyl-CoAs, albeit with differing efficiencies. Surprisingly, both CoA-SH and ATP were observed to inhibit the adenylation reaction at higher concentrations. Product quantification for kinetic determination was carried out by ESI-SIM-MS. Our results allow speculation as to evolutionary relationships within the large class of adenylate forming enzymes.     

An optimized ATP/PP(i)-exchange assay in 96-well format for screening of adenylation domains for applications in combinatorial biosynthesis

We report a new format for measuring ATP/[(32)P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower k(cat)/K(M). The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the combinatorial biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.     

The tetrameric structure of Haemophilus influenza hybrid Prx5 reveals interactions between electron donor and acceptor proteins

Cellular redox control is often mediated by oxidation and reduction of cysteine residues in the redox-sensitive proteins, where thioredoxin and glutaredoxin (Grx) play as electron donors for the oxidized proteins. Despite the importance of protein-protein interactions between the electron donor and acceptor proteins, there has been no structural information for the interaction of thioredoxin or Grx with natural target proteins. Here, we present the crystal structure of a novel Haemophilus influenza peroxiredoxin (Prx) hybrid Prx5 determined at 2.8-A resolution. The structure reveals that hybrid Prx5 forms a tightly associated tetramer where active sites of Prx and Grx domains of different monomers interact with each other. The Prx-Grx interface comprises specific charge interactions surrounded by weak interactions, providing insight into the target recognition mechanism of Grx. The tetrameric structure also exhibits a flexible active site and alternative Prx-Grx interactions, which appear to facilitate the electron transfer from Grx to Prx domain. Differences of electron donor binding surfaces in Prx proteins revealed by an analysis based on the structural information explain the electron donor specificities of various Prx proteins.     

单个活细胞中生物分子动态行为的FRET实时检测

分别采用两种不同绿色荧光蛋白(green fluorescent prote in,GFP)突变体作为荧光共振能量转移(fluo-rescence resonance energy transfer,FRET)对的供体和受体,并利用分子生物学技术将供体和受体分子分别与特定的生物分子融合,这种技术已经成为在单个活细胞中实时长时间检测蛋白质间的动态相互作用的主要技术。主要介绍了基于GFPs的FRET技术在单个活细胞中实时长时间研究生物分子动态行为的应用。     

Development of a baculovirus-based fluorescence resonance energy transfer assay for measuring protein-protein interaction.

A new baculovirus-based fluorescence resonance energy transfer (Bv-FRET) assay for measuring multimerization of cell surface molecules in living cells is described. It has been demonstrated that gonadotropin-releasing hormone receptor (GnRH-R) was capable of forming oligomeric complexes in the plasma membrane under normal physiological conditions. The mouse gonadotropin-releasing hormone receptor GnRH-R was used to evaluate the efficiency and potential applications of this assay. Two chimeric constructs of GnRH-R were made, one with green fluorescent protein as a donor fluorophore and the other with enhanced yellow fluorescent protein as an acceptor fluorophore. These chimeric constructs were coexpressed in an insect cell line (BTI Tn5 B1-4) using recombinant baculoviruses. Energy transfer occurred from the excited donor to the acceptor when they were in close proximity. The association of GnRH-R was demonstrated through FRET and the fluorescence observed using a Leica TSC-SPII confocal microscope. FRET was enhanced by the addition of a GnRH agonist but not by an antagonist. The Bv-FRET assay constitutes a highly efficient, reliable and convenient method for measuring protein-protein interaction as the baculovirus expression system is superior to other transfection-based methods. Additionally, the same insect cell line can be used routinely for expressing any recombinant proteins of interest, allowing various combinations of molecules to be tested in a rapid fashion for protein-protein interactions. The assay is a valuable tool not only for the screening of new molecules that interact with known bait molecules, but also for confirming interactions between other known molecules.     

Crystal structures of the tRNA:m2G6 methyltransferase Trm14/TrmN from two domains of life

Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNA(Phe) at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m(2)G6 (N(2)-methylguanosine) MTase (TTC)TrmN from Thermus thermophilus and its ortholog (Pf)Trm14 from Pyrococcus furiosus. Structures of (Pf)Trm14 were solved in complex with the methyl donor S-adenosyl-l-methionine (SAM or AdoMet), as well as the reaction product S-adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. (TTC)TrmN and (Pf)Trm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNA(Phe) of T. thermophilus and via site-directed mutagenesis.     

Crystal structure of GlcAT-S, a human glucuronyltransferase, involved in the biosynthesis of the HNK-1 carbohydrate epitope

The HNK-1 carbohydrate epitope is found in various neural cell adhesion molecules. Two glucuronyltransferases (GlcAT-P and GlcAT-S) are involved in the biosynthesis of HNK-1 carbohydrate. Our previous study on the crystal structure of GlcAT-P revealed the reaction and substrate recognition mechanisms of this enzyme. Comparative analyses of the enzymatic activities of GlcAT-S and GlcAT-P showed that there are notable differences in the acceptor substrate specificities of these enzymes. To elucidate differences between their specificities, we now solved the crystal structure of GlcAT-S. Residues interacting with UDP molecule, which is a part of the donor substrate, are highly conserved between GlcAT-P and GlcAT-S. On the other hand, there are some differences between these proteins in the manner they recognize their respective acceptor substrates. Phe245, one of the most important GlcAT-P residues for the recognition of acceptors, is a tryptophan in GlcAT-S. In addition, Val320, which is located on the C-terminal long loop of the neighboring molecule in the dimer and critical in the recognition of the acceptor sugar molecule by the GlcAT-P dimer, is an alanine in GlcAT-S. These differences play key roles in establishing the distinct specificity for the acceptor substrate by GlcAT-S, which is further supported by site-directed mutagenesis of GlcAT-S and a computer-aided model building of GlcAT-S/substrate complexes.     

Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions

A substantial range of protein-protein interactions can be readily monitored in real time using bioluminescence resonance energy transfer (BRET). The procedure involves heterologous coexpression of fusion proteins, which link proteins of interest to a bioluminescent donor enzyme or acceptor fluorophore. Energy transfer between these proteins is then detected. This protocol encompasses BRET1, BRET2 and the recently described eBRET, including selection of the donor, acceptor and substrate combination, fusion construct generation and validation, cell culture, fluorescence and luminescence detection, BRET detection and data analysis. The protocol is particularly suited to studying protein-protein interactions in live cells (adherent or in suspension), but cell extracts and purified proteins can also be used. Furthermore, although the procedure is illustrated with references to mammalian cell culture conditions, this protocol can be readily used for bacterial or plant studies. Once fusion proteins are generated and validated, the procedure typically takes 48-72 h depending on cell culture requirements.     

Annexin II is a major component of fusogenic endosomal vesicles

We have used an in vitro assay to follow the proteins transferred from a donor to an acceptor upon fusion of early endosomes. The acceptor was a purified early endosomal fraction immunoisolated on beads and the donor was a metabolically-labeled early endosomal fraction in suspension. In the assay, both fractions were mixed in the presence of unlabeled cytosol, and then the beads were retrieved and washed. The donor proteins transferred to the acceptor were identified by two- dimensional gel electrophoresis and autoradiography. Approximately 50 major proteins were transferred and this transfer fulfilled all criteria established for endosome fusion in vitro. However, only a small subset of proteins was efficiently transferred, if donor endosomes were briefly sonicated to generate small (0.1 micron diam) vesicles before the assay. These include two acidic membrane proteins, and three alkaline peripheral proteins exposed on the cytoplasmic face of the membrane. Partial sequencing and Western blotting indicated that one of the latter components is annexin II, a protein known to mediate membrane-membrane interactions. Immunogold labeling of cryosections confirmed that annexin II is present on early endosomes in vivo. These data demonstrate that annexin II, together with the other four proteins we have identified, is a major component of fusogenic endosomal vesicles, suggesting that these proteins are involved in the binding and/or fusion process.     

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a ''donor'' luciferase enzyme to an ''acceptor'' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.     

Two mutations in the HMG-box with very different structural consequences provide insights into the nature of binding to four-way junction DNA.

Mutation of the highly conserved tryptophan residue in the A-domain HMG-box of HMG1 largely, but not completely, destroys the protein tertiary structure and abolishes its supercoiling ability, but does not abolish structure-specific DNA binding to four-way junctions. Circular dichroism shows that the protein has some residual alpha-helix (< 10%) and does not re-fold in the presence of DNA. Structure-specific DNA binding might therefore be a property of some primary structure element, for example the N-terminal extended strand, which even in the unfolded protein would be held in a restricted conformation by two, largely trans, X-Pro peptide bonds. However, mutation of P5 or P8 of the A-domain to alanine does not abolish the formation of the (first) complex in a gel retardation assay, which probably arises from binding to the junction cross-over, although the P8 mutation does affect the formation of higher complexes which may arise from binding to the junction arms. Since mutation of P8 in the W49R mutant has no effect on structure-specific junction binding, we propose that some residual alpha-helix in the protein might be involved, implicating this element in the interactions of HMG-boxes generally with DNA.     

Homogeneous assay for biotin based on Aequorea victoria bioluminescence resonance energy transfer system

Here we describe a homogeneous assay for biotin based on bioluminescence resonance energy transfer (BRET) between aequorin and enhanced green fluorescent protein (EGFP). The fusions of aequorin with streptavidin (SAV) and EGFP with biotin carboxyl carrier protein (BCCP) were purified after expression of the corresponding genes in Escherichia coli cells. Association of SAV-aequorin and BCCP-EGFP fusions was followed by BRET between aequorin (donor) and EGFP (acceptor), resulting in significantly increasing 510 nm and decreasing 470 nm bioluminescence intensity. It was shown that free biotin inhibited BRET due to its competition with BCCP-EGFP for binding to SAV-aequorin. These properties were exploited to demonstrate competitive homogeneous BRET assay for biotin.     

On the enzyme system obtained from some mutants of Bacillus brevis deficient in gramicidin S formation

Twenty mutants of Bacillus brevis which were deficient in gramicidin S formation were isolated by N-methyl-N′-nitrosoguanidine treatment. In addition to three groups which have been previously classified, further two groups were established according to their characteristics of amino acid activating enzymes concerned with gramicidin S formation. The fourth group mutants had a phenylalanine activating enzyme, but they had an enzyme complex from which one specific enzyme among proline, valine and leucine activating enzymes was deleted. Some of them also the ability to form d-phenylalanyl-l-prolyl diketpiperazine (DKP) even though they had phenylalanine and proline activating enzymes. The fifth group mutants contained both a phenylalanine activating enzyme and a complex of prodine, valine, ornithine and leucine activating enzymes like as a wild strain, but did not synthesize gramicidin S, and also one of them could not form even DKP.Combination of enzymes from DKP (+) mutants of the fourth or fifth groups with the first group mutant which had an intact proline, valine, ornithine and leucine activating enzyme complex showed gramicidin S formation, but the combination of enzymes from DKP (−) mutants except a proline activating enzyme minus mutant with the first group mutant could not synthesize gramicidin S.