Substrate coordination to the active site Fe2+ of isopenicillin N synthase.

Orville, A.M.; Harpel, Mark R.; Chen, V.J.; Kriauciunas, Aidas; Fox, Brian G.; Srivastava, Kirti; Münck, Eckard; Lipscomb, John D.

doi:10.1016/0162-0134(91)84533-f

Cited by 4 publications

(10 citation statements)

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“…Our multiple-scattering analysis is best explained with two or three histidine imidazole ligands although we cannot exactly simulate the FT magnitude near 4.7 Á in Figure 4b. Although our EXAFS analysis cannot rigorously exclude the presence of a tyrosyl phenolate, we have previously argued against its involvement on the basis of other spectroscopic data (Chen et al, 1989;Orville et al, 1992).…”

Section: Discussionmentioning

confidence: 93%

“…ACV binding under anaerobic conditions unambiguously results in the incorporation of a sulfur atom into the Fe2+ coordination sphere. In the following paper (Orville et al, 1992), we provide evidence that the source of this coordinated thiolate is ACV rather than either of the two endogenous cysteines of IPNS.…”

mentioning

confidence: 83%

“…Two sets (A and B) of IPNS samples were prepared for XAS, each consisting of one sample of holoenzyme and one sample of IPNS-ACV complex which was generated by anaerobic addition of a solution of holoenzyme to solid ACV lyophilized from a pH 7.1 solution. ACV concentrations were chosen on the basis of its Kd value of 18 mM, estimated by Móssbauer titration (Orville et al, 1992), predicting 91% saturation at 0.19 M ACV. The ACV concentrations in the IPNS-ACV samples were estimated to be 0.10-0.19 M (set A) and 0.19 M (set B).…”

Section: Methodsmentioning

confidence: 99%

“…The binding of ACV to IPNS induces a number of spectral perturbations that are consistent with the coordination of an electron-rich atom to the metal center. These include the following: (a) significant decreases in the value of the Móssbauer isomer shift parameter, implying increased covalency of the iron center, upon formation of the IPNS-ACV and IPNS-ACV-NO complexes (Chen et al, 1989;Orville et al, 1992); (b) the appearance of a pink color and a visible absorption spectrum indicative of S -* iron charge transfer in IPNS-ACV-NO (Chen et al, 1989;Orville et al, 1992); and (c) the appearance of an absorption band at 385 nm characteristic of S -*• Cu2+ charge transfer upon adding ACV to Cu2+-reconstituted IPNS (Ming et al, 1990. While all of these changes may be explained by coordination of the ACV thiolate, the ligation of an endogenous cysteinyl thiolate or a tyrosine phenolate cannot be rigorously excluded.…”

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confidence: 99%

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X-ray absorption spectroscopic studies of the high-spin iron(II) active site of isopenicillin N synthase: evidence for iron-sulfur interaction in the enzyme-substrate complex

Scott

Wang²,

Eidsness³

et al. 1992

Biochemistry

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Isopenicillin N synthase from Cephalosporium acremonium (IPNS; M(r) 38.4K) is an Fe(2+)-requiring enzyme which catalyzes the oxidative conversion of (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N, with concomitant reduction of O2 to 2H2O. Chemical and spectroscopic data have suggested that catalysis proceeds via an enzyme complex of ACV bound to the iron through its cysteinyl thiolate [Baldwin, J. E., & Abraham, E. P. (1988) Nat. Prod. Rep. 5, 129-145; Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. Here we have employed the technique of Fe K-edge extended X-ray absorption fine structure (EXAFS) to characterize the iron site and to seek direct evidence for or against the formation of an Fe-S interaction upon ACV binding. Our data collected in the absence of substrate and O2 are consistent with the iron center of IPNS being coordinated by only (N,O)-containing ligands in an approximately octahedral arrangement and with an average Fe-(N,O) distance of 2.15 +/- 0.02 A. Upon anaerobic binding of ACV, the iron coordination environment changes considerably, and the associated Fe EXAFS cannot be adequately simulated without incorporating an Fe-S interaction at 2.34 +/- 0.02 A along with four or five Fe-(N,O) interactions at 2.15 +/- 0.02 A.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Discussionmentioning

confidence: 93%

mentioning

confidence: 83%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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X-ray absorption spectroscopic studies of the high-spin iron(II) active site of isopenicillin N synthase: evidence for iron-sulfur interaction in the enzyme-substrate complex

Scott

Wang²,

Eidsness³

et al. 1992

Biochemistry

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“…Results of experiments with [G-14C]shikimic acid, [U-14C]phenylalanine, and [U-14C]tyrosine show that tyrosine is by far the best precursor for erbstatin (74) in cultures of Streptomyces sp. MH435-hF3.49 Biosynthesis would seem to be similar to that of tuberin (75),50 but in the case of (74) further reaction occurs in which the chain undergoes a 1,2-shift concomitant with the introduction of the second hydroxy group.…”

Section: Erbstatinmentioning

confidence: 97%