Expression, purification, and characterization of HMWP2, a 229 kDa, six domain protein subunit of Yersiniabactin synthetase.
The six domain, 229 kDa HMWP2 subunit of the Yersinia pestis yersiniabactin (Ybt) synthetase has been expressed in soluble, full-length form in E. coli as a C-terminal His8 construct at low growth temperatures and with attenuated induction. All six domains of this nonribosomal peptide synthetase subunit, three phosphopantetheinylatable carrier protein domains (ArCP, PCP1, PCP2), one adenylation (A) domain, and two cyclization domains (Cy1, Cy2), have been assayed and are functional. Mutants that convert the phosphopantetheinylatable serine residue to alanine in each of the carrier protein domains accumulate acyl-S-enzyme intermediates upstream of the blocked apo carrier protein site. The ArCP mutant cannot be salicylated by the adenylation protein YbtE; the PCP1 mutant releases salicyl-cysteine from thiolysis of the Sal-S-ArCP intermediate; and the PCP2 mutant releases hydroxyphenyl-thiazolinyl-cysteine from the HPT-S-PCP1 acyl enzyme intermediate, all of which demonstrates processivity and directionality of chain growth. Restoration of the ArCP mutant's function was accomplished with the native ArCP fragment added in trans. The wild-type HMWP2 subunit accumulates hydroxyphenyl-4, 2-bithiazolinyl-S-enzyme at its most downstream PCP2 carrier site, presumably for transfer to the next subunit, HMWP1. The A domain was found to activate and transfer to PCP1 and PCP2 not only the natural L-Cys but also S-2-aminobutyrate, L-beta-chloroalanine, and L-Ser, enabling testing of the substrate specificity of the Cy domain. Probes of Cy domain function include mutagenesis of the Cy1 domain's conserved signature motif DX(4)DX(2)S to show that both D residues but not the S are crucial for both amide bond formation and heterocyclization. Also the Cy1 domain would accept an alternate upstream electrophilic donor substrate (2,3-dihydroxybenzoyl-S-ArCP) but would not process any of the three alternate downstream nucleophilic acceptors in place of Cys-S-PCP1, even for the amide bond-forming step in chain elongation.
Keating
T A
TA
Department of Biological Chemistry, Harvard Medical School, Boston, Massachusetts 02115, USA.
Miller
D A
DA
Walsh
C T
CT
D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis; D003066:Coenzyme A Ligases; D011756:Diphosphates / Q000378:metabolism; D005816:Genetic Complementation Test; D020035:Holoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006868:Hydrolysis; D033862:Iron-Binding Proteins; D007700:Kinetics; D008970:Molecular Weight; D010204:Pantetheine / Q000031:analogs & derivatives / Q000378:metabolism; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D033902:Periplasmic Binding Proteins; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D020156:Salicylic Acid / Q000378:metabolism; D013379:Substrate Specificity; D015010:Yersinia pestis / Q000201:enzymology* / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D011756:Diphosphates; D020035:Holoenzymes; D033862:Iron-Binding Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins; D010204:Pantetheine; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; C408477:YbtE protein, Yersinia pestis; D010453:Peptide Synthases; C003129:4'-phosphopantetheine; D020156:Salicylic Acid