1hb1: Difference between revisions
No edit summary |
No edit summary |
||
| Line 4: | Line 4: | ||
==Overview== | ==Overview== | ||
BACKGROUND: Isopenicillin N synthase (IPNS) catalyses formation of | BACKGROUND: Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the beta-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring. RESULTS: Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme-substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed. CONCLUSIONS: In the absence of its natural reaction partner (the N-H proton of the L-cysteinyl-D-valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding beta-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate. | ||
==About this Structure== | ==About this Structure== | ||
| Line 13: | Line 13: | ||
[[Category: Emericella nidulans]] | [[Category: Emericella nidulans]] | ||
[[Category: Single protein]] | [[Category: Single protein]] | ||
[[Category: Adlington, R | [[Category: Adlington, R M.]] | ||
[[Category: Baldwin, J | [[Category: Baldwin, J E.]] | ||
[[Category: Burzlaff, N | [[Category: Burzlaff, N I.]] | ||
[[Category: Clifton, I | [[Category: Clifton, I J.]] | ||
[[Category: Elkins, J | [[Category: Elkins, J M.]] | ||
[[Category: Ogle, J | [[Category: Ogle, J M.]] | ||
[[Category: Roach, P | [[Category: Roach, P L.]] | ||
[[Category: Rutledge, P | [[Category: Rutledge, P J.]] | ||
[[Category: FE2]] | [[Category: FE2]] | ||
[[Category: OCV]] | [[Category: OCV]] | ||
| Line 29: | Line 29: | ||
[[Category: penicillin biosynthesis]] | [[Category: penicillin biosynthesis]] | ||
''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 12:59:23 2008'' | ||
Revision as of 13:59, 21 February 2008
|
ISOPENICILLIN N SYNTHASE FROM ASPERGILLUS NIDULANS (ANAEROBIC ACOV FE COMPLEX)
OverviewOverview
BACKGROUND: Isopenicillin N synthase (IPNS) catalyses formation of bicyclic isopenicillin N, precursor to all penicillin and cephalosporin antibiotics, from the linear tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine. IPNS is a non-haem iron(II)-dependent enzyme which utilises the full oxidising potential of molecular oxygen in catalysing the bicyclisation reaction. The reaction mechanism is believed to involve initial formation of the beta-lactam ring (via a thioaldehyde intermediate) to give an iron(IV)-oxo species, which then mediates closure of the 5-membered thiazolidine ring. RESULTS: Here we report experiments employing time-resolved crystallography to observe turnover of an isosteric substrate analogue designed to intercept the catalytic pathway at an early stage. Reaction in the crystalline enzyme-substrate complex was initiated by the application of high-pressure oxygen, and subsequent flash freezing allowed an oxygenated product to be trapped, bound at the iron centre. A mechanism for formation of the observed thiocarboxylate product is proposed. CONCLUSIONS: In the absence of its natural reaction partner (the N-H proton of the L-cysteinyl-D-valine amide bond), the proposed hydroperoxide intermediate appears to attack the putative thioaldehyde species directly. These results shed light on the events preceding beta-lactam closure in the IPNS reaction cycle, and enhance our understanding of the mechanism for reaction of the enzyme with its natural substrate.
About this StructureAbout this Structure
1HB1 is a Single protein structure of sequence from Emericella nidulans with , and as ligands. Known structural/functional Site: . Full crystallographic information is available from OCA.
ReferenceReference
Alternative oxidation by isopenicillin N synthase observed by X-ray diffraction., Ogle JM, Clifton IJ, Rutledge PJ, Elkins JM, Burzlaff NI, Adlington RM, Roach PL, Baldwin JE, Chem Biol. 2001 Dec;8(12):1231-7. PMID:11755401
Page seeded by OCA on Thu Feb 21 12:59:23 2008