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Three Conserved Glycine Residues in Valine Activation of Gramicidin S Synthetase 2 from Bacillus Brevis1

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Three Conserved Glycine Residues in Valine Activation of Gramicidin S Synthetase 2 from Bacillus Brevis1 J. Biochem. 117, 276-282 (1995) Three Conserved Glycine Residues in Valine Activation of Gramicidin S Synthetase 2 from Bacillus brevis1 Masaki Saito, Kazuko Hori, Toshitsugu Kurotsu, Masayuki Kanda, and Yoshitaka Saito Department of Biochemistry, Hyogo College of Medicine, Mukogawa-cho, Nishinomiya, Hyogo 663 Received for publication, July 27, 1994 The translated product from the gene fragment containing the second and third domains of gramicidin S synthetase 2 was purified to an essentially homogeneous state. It showed valine and ornithine-activating activity and the second domain was proved to be the valine-activating domain. Three mutant genes from Bacillus brevis Nagano, BI-3, E-4, and E-5 strains, which encode defective valine-activating domains of gramicidin S synthetase 2, were sequenced. By comparison with the wild-type gene, single point mutations of guanine to adenine were found at the three conserved glycine codons; the 5303rd guanine in BI-3, the 5378th guanine in E-4, and the 4967th guanine in E-5, which corresponded to codon changes of the 1768th glycine to glutamic acid and the 1793rd and the 1656th glycine to aspartic acid. Loss of valine-adenylation activity by mutation at the 1656th glycine proved the direct participation of the TSGT/STGXPKG motif in the adenylation reaction, and suggests that this glycine residue with the conserved lysine residue of the motif forms the phosphate-binding loop for ATP-binding. The 1793rd glycine is a member of the YGXTE motif which was also conserved among adenylate-forming enzymes except acetyl-CoA synthetases. The 1768th glycine residue appears to maintain the conformation of the active site for aminoacyl adenylation since this residue is retained among the adenylate-forming enzymes, though flanking regions are not conserved. These results suggest that these glycine residues are essential for adenylate formation in the antibiotic peptide synthetase family and some other adenylate-forming enzymes. Key words: aminoacyl adenylation, ATP-binding, Bacillus brevis, gramicidin S synthe tase 2, peptide synthetase. A cyclic peptide antibiotic, gramicidin S (D-Phe L-Pro theine (8), and the two identical pentapeptides cyclize to L-Val-L-Orn-L-Leu)2 is produced nonribosomally by two form gramicidin S by an unknown mechanism. complementary multienzymes: gramicidin S synthetase 1 To clarify the reaction mechanism of such complicated (GS1) [EC 5.1.1.11] and 2 (GS2) (1). GS1, with a enzymes, we cloned and sequenced the entire nucleotide molecular weight of 120,000 activates, thioesterifies, and sequences of gramicidin S synthetase 1 and 2 genes (grsl racemizes phenylalanine, the first amino acid of gramicidin and grs2) (9-11). They were organized in an operon and S (2), and GS2 with a molecular weight of 510,000 acti had three open reading frames in the following order, grsT, vates and thioesterifies the other constituent amino acids, grsl, and grs2. The grsT gene encoded a protein of 256 proline, valine, ornithine, and leucine (3, 4). Individual amino acids homologous to fatty acid thioesterases. The amino acids are activated in a similar reaction to that grsl gene encoded a protein of 1,098 amino acids and the catalyzed by aminoacyl-tRNA synthetase (5), grs2 gene encoded a protein of 4,450 amino acids which consisted of four repeated domains of about 1,050 amino amino acid+ATP+Enz_??_aminoacyl AMP-Enz F PPi acids. The grsl protein and individual domains of the grs2 then the activated amino acids are transferred to enzyme protein had conserved 600-amino-acid sequences which sulfhydryl groups to form the aminoacyl-S-enzyme com were thought to be the activation sites for each of the plex (6), constituent amino acids of gramicidin S. Recently, several peptide synthetase genes have been cloned and sequenced aminoacyl AMP-Enz aminoacyl-S-Enz+AMP from bacteria and fungi (12-23). These proteins also had The synthesis of gramicidin S is started by a transfer of the conserved and repeated sequence of about 600 amino D-phenylalanine from GS1 to GS2 (7). The subsequent acids. The first, the third, and the fourth domains of grs2 polymerization process is followed by sequential thiolation protein were proved to be proline-, ornithine-, and leucine and transpeptidation reactions through 4•L-phosphopante activating domains, respectively (10, 11). In this paper, we report that the second domain of grs2 1 This work was supported in part by a research grant from Hyogo College of Medicine. protein is related to valine activation and that three Abbreviations: p-amidino PMSF, (p-amidinophenyl)methanesulfo conserved glycine residues of the valine-activating domain nyl fluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel are essential for adenylate formation among aminoacyl and electrophoresis. acyl adenylate-forming enzymes. 276 J. Biochem. Aminoacyl Adenylation Site of Gramicidin S Synthetase 2 277 Fig. 1. Diagram of gramicidin S biosynthetic genes and construction of the expression vector containing the second and third domains of grs2 gene. The open box indicates the entire grs2 operon. pGS3KX, pGS3KX-S, and pGS2-7 were obtained by subcloning for sequencing (11). The recombinant plas mid pGS3KX19XX which expresses the second and third domains of grs2 gene, was constructed in pUC 19 by ligating the Xbal fragment of pGS2-7 with an insert of pGS3KX. Numerals under the box are nucleotide numbers of the grs2 gene from the deduced ATG initiation triplet. were determined by direct sequencing of the genomic DNA. MATERIALS AND METHODS The chromosomal DNA preparations from the wild and mutant strains of B. brevis were obtained as described Bacterial Strains and Plasmids•\A gramicidin S-pro previously (9) and used as templates. Two overlapped ducing strain of Bacillus brevis Nagano and its mutant fragments of grs2-val gene were amplified using two sets of stains, BI-3, E-4, and E-5 were used as sources of forward and reverse primers (24mers), 5•L-AGTACTTG chromosomal DNA (24, 25). Escherichia coli JM103(zPl) GGATTTCTCAGATCGG-3•L (primer 1; forward strand), [4(lac-pro), thi, strA, supE, endAsbcB, hsdR, F•LtraD36, 5•L-CTGTTGGGTAAGCATAATGCGTAC-3•L (primer 2; proAB, laclPZ4M15] and E. coli DH-1 [recA1, endAl, reverse strand), 5•L-TATCAGAAATCGAGATATTGTC gyrA96, thi-1, hsdRl7 (rk-, mk-), supE44] were used as TG-3•L (primer 3; forward strand), and 5•L-GCCCCTTC host cells. The plasmid vectors, pUC18 and pUC19, were AAATTCATAAAGGATG-3•L (primer 4; reverse strand). used for cloning or construction of recombinant plasmids. These primers annealed the nucleotides nt 2958 to nt 2981 The recombinant plasmids pGS3KX and pGS2-7 were (primer 1), nt 4810 to nt 4833 (primer 2), nt 4430 to nt obtained by subcloning for the sequencing of the grs2 gene 4453 (primer 3), and nt 6321 to 6344 (primer 4). The DNA as described previously (11). These inserts encoded a part sequences were determined by the dideoxy-nucleotide of the second domain and the full first or third domains of chain termination method of Sanger et al. (27). Sequenc the grs2 protein (Fig. 1). The recombinant plasmid, ing and analysis were carried out as described previously pGS3KX19XX, which expresses the second and the third (26). domains of the grs2 gene, was constructed in pUC19 by Biochemicals•\32PPi was purchased from the Radio ligating the pGS3KX insert with the smallest Xbal frag chemical Centre (Amersham, UK). Restriction endonucle ment of pGS2-7. The direction of the Xbal fragment in the ases were purchased from Takara Shuzo (Kyoto) and recombinant plasmid was confirmed by sequencing. Nippon Gene (Toyama). Gene AmpTM PCR reagent kit, Analysis and Purification of the Translated Product Ampli TagTM DNA polymerase, DNA ligation kit, and E. coli alkaline phosphatase were purchased from Takara from the Recombinant Plasmid pGS3KX19XX•\The trans lated product of the recombinant plasmid, pGS3KX19XX, Shuzo. DNase and RNase were obtained from Sigma was prepared on a large scale from the transformed E. coli Chemical (St. Louis, USA). All other chemicals were JM103LJP1 cells as described previously (9-11). An aliquot standard commercial products. of the ammonium sulfate fraction was separated by gel filtration FPLC on Superose 12 (Pharmacia) equilibrated RESULTS with buffer A [20mM sodium phosphate buffer (pH 7.5) containing 150mM NaCl, 1mM dithiothreitol, 1mM Identification of the Second Domain of Grs2 Gene as the Valine-Activating Domain The recombinant plasmid, MgC12i 0.5mM EDTA, 10 pM p-amidino PMSF, and 10% pGS3KX19XX, was constructed to express the full second glycerol] (11). Fraction numbers 24 to 28 from two-cycle and third domains of the grs2 protein (Fig. 1). The translat elution were pooled and applied to a Mono Q column ed product of pGS3KX19XX was partially purified by 50% equilibrated with buffer B [50mM Tris-HCl buffer (pH saturated ammonium sulfate fractionation from the lysate 8.0) containing 1mM dithiothreitol and 10% glycerol] and of the transformed cells as described previously (9, 10) and eluted with a linear gradient of 0 to 0.5 M NaCl in the same further purified by gel filtration FPLC on Superose 12 and buffer. Each fraction from Superose 12 and Mono Q column ion exchange FPLC on Mono Q. chromatography was used for assay of the amino acid The translated products of pGS3KX19XX had valine dependent ATP-32M exchange activities and for immuno and ornithine-dependent exchange activities. In the Super blot analysis. Immunoreactive proteins to GS2 antibodies ose 12 chromatography, these activities were eluted at the were analyzed by dot blotting and Western blotting as described previously (10, 26). Activities of the amino acid position corresponding to a protein of about 300kDa, which agreed with the deduced size of the expressed protein of the dependent ATP-32PP; exchange reaction were measured as insert. These fractions showed immunoreactivity to GS2 described previously (3).
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