Codon optimized nucleotide sequence for GeoM

1 ATGAACGAAA TCGTGGAAAA TAACCTGATG GAAATCATCA AAGATAAACT GCTGTACCCG 61 AGCGAAAAAA AAAAAGAAAT CGAAAACAAC GTGCCGATTA ACGAGCTGCT GTTCCAAGAA 121 AAAACCAACA AATGGATCGA CCTGTTCTAT CAGTATCTGA ACGTGTGCAA AAACAACATT 181 GAAGTGGATC AGCTGTATAG CAACGAAGAA AACCTGAGCA ATCCGTTTAG CAGCTTTTTC 241 AAAGAATTTC TGGAAATTGC GAGCCGTTAT CTGGATAGCA GCCTGAAAAG CCAGGTGAAC 301 AACTATGATG ATCTGAGCCG TATTTGCAAC CTGACCAGCA TCAAAAATGA TATCATGAAA 361 TTCATCCACG AAAACCTGGT GTATGTGAGC ATTCGTACCC TGATTCAGGA TCTGAATGAA 421 GAACGTGAAA AAGGCAACCT GATTGGCGAT ACCCCGAAAG AACGCTATAA CTTCTACGTG 481 GATCAGATTC TGAGCAGCCC GGATAAAAAA TTCGAGCTGA TCAAAAAATA TCCGGTGCTG 541 GTGCGTATTC TGATTGAATT TATCCTGAAC AAAATCGATA GCATCGTGGA AAGCATTTAC 601 CGCTTTCTGA AAGATCGTAG CGAGCTGGTG CATATTTTCC ACCTGAGCGA TGATGATATT 661 CTGACCAGCC TGACTCTGCA GAGCGGCGAT AGCCATAACA ACGGCCGTAG CGTGATCATT 721 TTTCAGTTCA GCAGCAAAAA AAAAATCGTG TACAAACCGC GTAGCCTGAG CATTGATCTG 781 CATTTTCAGC AGTTTCTGGA ATGGATCAAC GGCAAAAAAC CGAGCCTGCA GCTGAAAACG 841 ATTACCATTC TGAACAAAGA TCAGTATGGC TGGCAGGAAT TTGTTGAATA TAAACCGTGC 901 AGCTCTAACA ACGAGCTGTC TCGTTTTTAT GAACGTCAGG GCAACTATAT TGCGATTCTG 961 TATATTCTGA ACGCGACCGA TTTTCATTTT GAAAACCTGA TTGCGAACGG CGAACACCCG 1021 GTGCTGATTG ATCTGGAAGG CCTGGTGCAG AACACCGTGA AACTGCCGCG TAAAGCGAGC 1081 AGCGCGTATG ATATTGCGTT TAGCAAACTG ACCGATAGCG TTCTGAGCAC CGGCATGCTG 1141 CCGGCGACCT TTATGCAGGC GAATATCTAT GATTTTGATC TGAGCGGCCT GGGCGGTGAT 1201 GAAGGTCAGC CGACCGGCCT GGAAACCTTT ACCATTGAAA ATCCGCTGAC CGATGAAATG 1261 CGTGTGATTA AAGTGCCGGC GTTTAGCCAG AGCAGCAAAA ACAAACCGTA TCTGAAAGAA 1321 GAAAAAGAAA TTGCGGTGAC CGATTATAGC AGCGAAATCA TTAAAGGCTT CCGCGAAATG 1381 TATACCCTGC TGCTGCATAA CAAAAAGGAG CTGCTGTCTC AGAACGGCCC GATTTACCTG 1441 TTTAAAGGCG ATAAAGTGCG TATTATTCTG CGTAGCACCC AGGTGTATAG CACCTTCCTG 1501 GATAGCAGCT TTCATCCGGA TTATCTGAAA GATGGCTATG AACGTGAACG CCTGATTAAC 1561 TTTCTGTGGA TCGGCAAAGA AAACCATCCG GAATATCAGG ATGCGATCAT GTATGAATGC 1621 CGCGATATTC TGAATGGCGA TATCCCGTAT TTCTATTGCT ATACCGATAG CAGCGATCTG 1681 TATCATCCGA TTGGCATCGT GAAACACAAC TTTTTCTATG AAAGCAGCTT TAACAGCCTG 1741 CTGGAAAAAG TGAAAATGAT CAACGAAGAA GATCTGAATT TCCAGATCGA AATTCTGACC 1801 AATAGCCTGC TGGCCCAGTA TAGCAACAAA ACCCATAGCC ATGCGAACGT GAGCAACCGT 1861 GTGTATAACC TGGATAAAAT CAGCGGCAAC TTTCGTCGTG AATCTTTTCT GGAAGTGAGC 1921 GAAAAAATTG CGGATAACAT CAAAGAAAAC GCGATCTTCG GTAAAGAAAA CGATGTGACC 1981 TGGCTGGGCC TGAACCTGAC CATTGAAGAT AAATGGACCT TCAAACCGCT GGATTTCGAT 2041 CTGTATGATG GCGTGCTGGG CATTGGCCTG TTTTATGCGA ACCTGTATAA CCTGAACAAA 2101 CGCAAAGAAT ACAAAATCCT GGCCGAAAAA ACCGTGCAGA CCGCGCTGAA CTATCTGGAA 2161 TATTATCCGG CGAAACTGCC GCTGTCTGCG TTTTATGGCT ATGGCGCGTA TGCGTATGTG 2221 CTGGGCAACT TTAGCATCAT CTTTAATAAC AGCGAATACC TGACCTATGT GAAAAAAGTG 2281 CTGAACAAAG CGGCGAACAC CATCGAAGAT GATCAGCTGC TGGATTTTCT GGGCGGTGCG 2341 GCGGGTCTGA TTATTGTGTG CATCCACCTG TATAAAAAAA CCGGCGAACA GTATCTGCTG 2401 GATATTGCGA ACAAATGCGG TGAGCTGCTG CTGCGTAAAA AAGAAAAACA GGCCATGGGC 2461 ATTGGCTGGC GTCCGAACCA GGTGAATAAA CCGCTGGCCG GTCTGGCCCA TGGTAGCAGC 2521 GGCTTTACCT GGGCGCTGAT GGCGCTGTAT AAATTCACCA AAAACTACAA CTATAAAGAA

2641 CTGGTTCTGA GCGACGATGG CGATTATTAT GGCAACAGCA TGTGGTGCCA TGGTGCGGCC 2701 GGTATTGGCA TGAGCCGTAT CATGATGAGC GAATACTATA ACAGCCACGA TCTGAAAAAC 2761 GATATTGAAA TCGCGATTCG TCAGACCCTG AAAAGCGGCT TTGGCGGCAC CCATTGCCTG 2821 TGCCATGGCG ATCTGGGCAA CCTGGACCTG TTTCTGCTGG CCTCTAGCAA ATTTAAAAAC 2881 GAAGAAATGA AAGAAACCGC GCTGAAAATT GGCGAATATA TCGCGAGCGA TATTACCTAT 2941 GGCAACCTGA AATATGGCAC CCCGAGCGGC ACCAAAAATC TGGGTCTGAT GCTGGGCCAG 3001 GCGGGTATTG GCTATGGCTT TCTGCGTCTG GCCTATCCGG AAATTGTGCC GAGCGTGCTG 3061 ACTCTGCAGC TGAACCATTA A

4.8 References

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32. Kodani, S., Lodato, M. A., Durrant, M. C., Picart, F., and Willey, J. M. (2005) SapT, a lanthionine-containing peptide involved in aerial hyphae formation in the streptomycetes, Mol Microbiol 58, 1368-1380.

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Appendix: Structural characterization of lantibiotic dehydrogenase ElxO1

A.1 Introduction

The lantibiotic epilancin 15X is a member of the epilancin-group of peptides and is produced by Staphylococcus epidermidis 15X154 (1-2). S. epidermidis 15X154, a clinical strain isolated from a wound infection, is a colonizer of the human skin (3). Other staphylococcal lantibiotics include Pep5 (4), epidermin (5), epicidin 280 (6), and epilancin K7 (7-8). Epilancin 15X was shown to have potent antimicrobial activity against pathogenic bacteria, including MRSA and VRE (9). Epilancin 15X contains ten post-translationally modified amino acids, one lanthionine, two methyllanthionine bridges, and an unusual N-terminal 2-hydroxypropionyl group (D-lactate, Figure 1.1 and 1.3, Chapter 1). The gene cluster and biosynthetic pathway of

epilancin 15X was recently characterized (2). The N-terminal lactate was shown to be produced by dehydration of the first serine residue in the core peptide by the lantibiotic dehydratase ElxB, followed by proteolytic removal of the leader peptide by the lantibiotic protease ElxP. The spontaneous hydrolysis of the N-terminal dehydroalanine to a pyruvate group and reduction of the newly generated pyruvate to lactate by the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent oxidoreductase ElxO complete the biosynthesis of epilancin 15X. The N- terminal lactate group was shown to impart stability to epilancin 15X against bacterial aminopeptidases. Three lantibiotics namely, epilancin K7 (7-8), SWLP1 (10), and epicidin 280 (6) have also been reported to contain an N-terminal lactate group. The biosynthetic gene cluster of epicidin 280 contains a homologous oxidoreductase EciO and was suggested to be involved in the biosynthesis of its N-terminal lactate (6). The enzymatic activity of ElxO has been reconstituted in vitro and ElxO was shown to have relaxed substrate specificity by Dr. Juan Esteban Velásquez, as various N-terminal ketone-containing peptides were tolerated as

substrates. The N-terminal lactate group generated by ElxO can be engineered into other lantibiotics in order to enhance their biological stability. In this study, the crystal structure of ElxO was determined providing insights into the catalytic mechanism.

A.2 Experimental procedures