Structure of the heme biosynthetic Pseudomonas aeruginosa porphobilinogen synthase in complex with the antibiotic alaremycin.
The recently discovered antibacterial compound alaremycin, produced by Streptomyces sp. A012304, structurally closely resembles 5-aminolevulinic acid, the substrate of porphobilinogen synthase. During the initial steps of heme biosynthesis, two molecules of 5-aminolevulinic acid are asymmetrically condensed to porphobilinogen. Alaremycin was found to efficiently inhibit the growth of both Gram-negative and Gram-positive bacteria. Using the newly created heme-permeable strain Escherichia coli CSA1, we are able to uncouple heme biosynthesis from bacterial growth and demonstrate that alaremycin targets the heme biosynthetic pathway. Further studies focused on the activity of alaremycin against the opportunistic pathogenic bacterium Pseudomonas aeruginosa. The MIC of alaremycin was determined to be 12 mM. Alaremycin was identified as a direct inhibitor of recombinant purified P. aeruginosa porphobilinogen synthase and had a K(i) of 1.33 mM. To understand the molecular basis of alaremycin's antibiotic activity at the atomic level, the P. aeruginosa porphobilinogen synthase was cocrystallized with the alaremycin. At 1.75-A resolution, the crystal structure reveals that the antibiotic efficiently blocks the active site of porphobilinogen synthase. The antibiotic binds as a reduced derivative of 5-acetamido-4-oxo-5-hexenoic acid. The corresponding methyl group is, however, not coordinated by any amino acid residues of the active site, excluding its functional relevance for alaremycin inhibition. Alaremycin is covalently bound by the catalytically important active-site lysine residue 260 and is tightly coordinated by several active-site amino acids. Our data provide a solid structural basis to further improve the activity of alaremycin for rational drug design. Potential approaches are discussed.
Heinemann
Ilka U
IU
Institute of Microbiology, Technical University Braunschweig, Spielmannstrasse 7, Braunschweig D-38106, Germany.
Schulz
Claudia
C
Schubert
Wolf-Dieter
WD
Heinz
Dirk W
DW
Wang
Yang-G
YG
Kobayashi
Yuichi
Y
Awa
Yuuki
Y
Wachi
Masaaki
M
Jahn
Dieter
D
Jahn
Martina
M
D000614:Aminocaproates / Q000494:pharmacology*; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001419:Bacteria / Q000187:drug effects; D001426:Bacterial Proteins / Q000096:biosynthesis; D003460:Crystallization; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005822:Genetic Vectors; D006090:Gram-Negative Bacteria / Q000187:drug effects; D006094:Gram-Positive Bacteria / Q000187:drug effects; D006418:Heme / Q000096:biosynthesis*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D017021:Methanosarcina barkeri / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D008826:Microbial Sensitivity Tests; D008958:Models, Molecular; D000623:Porphobilinogen Synthase / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D011487:Protein Conformation; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D015032:Zinc / Q000494:pharmacology
D000614:Aminocaproates; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C506902:alaremycin; D006418:Heme; D000623:Porphobilinogen Synthase; D008274:Magnesium; D015032:Zinc