Structural Studies on the Reaction of Isopenicillin N Synthase with the Truncated Substrate Analogues -(L-
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- 作者：Alexandra J. Long ; Ian J. Clifton ; Peter L. Roach ; Jack E. Baldwin ; Peter J. Rutledge ; and Christopher J. Schofield
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：May 3, 2005
- 年：2005
- 卷：44
- 期：17
- 页码：6619 - 6628
- 全文大小：375K
- 年卷期：v.44,no.17(May 3, 2005)
- ISSN：1520-4995
文摘
Isopenicillin N synthase (IPNS), a non-heme iron(II)-dependent oxidase, catalyzes conversionof the tripeptide 
ta.gif" BORDER=0 >-(L-
-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to bicyclic isopenicillin N (IPN),concomitant with the reduction of dioxygen to two molecules of water. Incubation of the "truncated"substrate analogues ta.gif" BORDER=0 >-(L--aminoadipoyl)-L-cysteinyl-glycine (ACG) and ta.gif" BORDER=0 >-(L--aminoadipoyl)-L-cysteinyl-D-alanine (ACA) with IPNS has previously been shown to afford acyclic products, in which the substratecysteinyl residue has undergone a two-electron oxidation. We report X-ray crystal structures for theanaerobic IPNS/Fe(II)/ACG and IPNS/Fe(II)/ACA complexes, both in the absence and presence of thedioxygen analogue nitric oxide. The overall protein structures are very similar to those of the correspondingIPNS/Fe(II)/ACV complexes; however, significant differences are apparent in the vicinity of the activesite iron. The structure of the IPNS/Fe(II)/ACG complex reveals that the C-terminal carboxylate of thissubstrate is oriented toward the active site iron atom, apparently hydrogen-bonded to an additional waterligand at the metal; this is a different binding mode to that observed in the IPNS/Fe(II)/ACV complex.ACA binds to the metal in a manner that is intermediate between those observed for ACV and ACG. Theaddition of NO to these complexes initiates conformational changes such that both the IPNS/Fe(II)/ACG/NO and IPNS/Fe(II)/ACA/NO structures closely resemble the IPNS/Fe(II)/ACV/NO complex. These resultsfurther demonstrate the feasibility of metal-centered rearrangements in catalysis by non-heme iron enzymesand provide insight into the delicate balance between hydrophilic-hydrophobic interactions and stericeffects in the IPNS active site.
ta.gif" BORDER=0 >-(L--aminoadipoyl)-L-cysteinyl-D-valine (ACV) to bicyclic isopenicillin N (IPN),concomitant with the reduction of dioxygen to two molecules of water. Incubation of the "truncated"substrate analogues ta.gif" BORDER=0 >-(L--aminoadipoyl)-L-cysteinyl-glycine (ACG) and ta.gif" BORDER=0 >-(L--aminoadipoyl)-L-cysteinyl-D-alanine (ACA) with IPNS has previously been shown to afford acyclic products, in which the substratecysteinyl residue has undergone a two-electron oxidation. We report X-ray crystal structures for theanaerobic IPNS/Fe(II)/ACG and IPNS/Fe(II)/ACA complexes, both in the absence and presence of thedioxygen analogue nitric oxide. The overall protein structures are very similar to those of the correspondingIPNS/Fe(II)/ACV complexes; however, significant differences are apparent in the vicinity of the activesite iron. The structure of the IPNS/Fe(II)/ACG complex reveals that the C-terminal carboxylate of thissubstrate is oriented toward the active site iron atom, apparently hydrogen-bonded to an additional waterligand at the metal; this is a different binding mode to that observed in the IPNS/Fe(II)/ACV complex.ACA binds to the metal in a manner that is intermediate between those observed for ACV and ACG. Theaddition of NO to these complexes initiates conformational changes such that both the IPNS/Fe(II)/ACG/NO and IPNS/Fe(II)/ACA/NO structures closely resemble the IPNS/Fe(II)/ACV/NO complex. These resultsfurther demonstrate the feasibility of metal-centered rearrangements in catalysis by non-heme iron enzymesand provide insight into the delicate balance between hydrophilic-hydrophobic interactions and stericeffects in the IPNS active site.