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. 2004 Sep 1;382(Pt 2):659-66.
doi: 10.1042/BJ20040529.

Active-site-mediated elimination of hydrogen fluoride from a fluorinated substrate analogue by isopenicillin N synthase

Annaleise R Grummitt  1 , Peter J Rutledge, Ian J Clifton, Jack E Baldwin
Affiliations

Active-site-mediated elimination of hydrogen fluoride from a fluorinated substrate analogue by isopenicillin N synthase

Annaleise R Grummitt et al. Biochem J. .

Abstract

Isopenicillin N synthase (IPNS) is a non-haem iron oxidase that catalyses the formation of bicyclic isopenicillin N from delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine (ACV). In this study we report a novel activity for the iron of the IPNS active site, which behaves as a Lewis acid to catalyse the elimination of HF from the fluorinated substrate analogue, delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-beta-fluorovaline (ACbetaFV). X-Ray crystallographic studies of IPNS crystals grown anaerobically with ACbetaFV reveal that the valinyl beta-fluorine is missing from the active site region, and suggest the presence of the unsaturated tripeptide delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-isodehydrovaline in place of substrate ACbetaFV. (19)F NMR studies confirm the release of fluoride from ACbetaFV in the presence of the active IPNS enzyme. These results suggest a new mode of reactivity for the IPNS iron centre, a mechanism of action that has not previously been reported for any of the iron oxidase enzymes.

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Figures

Scheme 1
Scheme 1. Proposed mechanism of action of IPNS in reaction with its natural substrate, ACV
Scheme 2
Scheme 2. Synthetic route to ACβFV 3
(i) BOC2O, NaOH, tBuOH, H2O, 98%; (ii) p-ClCH2(C6H4)OCH3, Et3N, DMF, 99%; (iii) 1, iso-butylchloroformate, Et3N, THF, 2, S-benzhydryl-L-cysteine, Et3N, H2O, 71%; (iv) (CH3)2O, nBuLi, THF, −60 °C, quantitative; (v) DAST, DCM, −30 °C, 80%; (vi) HCl (aq), Δ; (vii) TsOH, H2O; (viii) Ph2CN2, CH3CN, Et2O; (ix) TsOH, H2O, 10% (over 4 steps); (x) EDCI, HOBt, Et3N, DCM, 87%; (xi) TFA (1), anisole; HPLC (2), 70%.
Figure 1
Figure 1. X-ray crystal structure of the complex formed from anaerobic interaction of ACβFV 3 with IPNS, to 1.30 Å resolution
(A) The two substrate orientations are shown separately in light and dark grey with a 7:3 occupancy ratio respectively. (B) The isopropyl region of ACβFV-derived product (light grey) overlaid with ACV (dark grey) for comparison (note the planarity in the electron density about the β-carbon of the ACβFV-derived structure). The 2mFo-DFc electron density map is contoured at 1.0σ.
Figure 2
Figure 2. X-ray crystal structure of complex arising from anaerobic ACβFV–IPNS interaction, showing relative torsion angles between isopropyl group and cysteinyl carbonyl for conformations (A) and (B) (torsion angles indicated)
Figure 3
Figure 3. 19F{1H} NMR spectra (2H2O, 235 MHz) resulting from anaerobic incubation of ACβFV 3 with (a) active IPNS, (b) boiled IPNS, and (c) no IPNS, under crystallization conditions [ACβFV 3.1 mg/ml, FeSO4 2.1 mM, Li2SO4 1.0 M, Tris/HCl 76 mM, (IPNS 27 mg/ml), pH 8.5, 17 °C, 3 days]
Scheme 3
Scheme 3. Alternative products obtainable by elimination of HF from ACβFV 3
See this image and copyright information in PMC

References

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