Crystal Structure of Hemoprotein Domain of P450BM-3, a Prototype for Microsomal P450's

Ravichandran, K. G.; Boddupalli, Sekhar; Hasermann, Charles A.; Peterson, Julian A.; Deisenhofer, Johann

doi:10.1126/science.8342039

Cited by 912 publications

(711 citation statements)

References 33 publications

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“…A crystallographic structure has not been determined for any wild-type mammalian P450. However, a crystallographic structure of the haemoprotein domain of P450 BM-$ , which is considered to be functionally related to microsomal P450s, has been determined at 2.0 A H resolution [26]. The structural similarity of P450 BM-$ to eicosanoid-synthesizing P450s, PGIS and TXAS is reflected in their 25 % sequence identity and the substrate similarity.…”

Section: Discussionmentioning

confidence: 99%

Substrate access channel topology in membrane-bound prostacyclin synthase

Deng¹,

Huang²,

So³

et al. 2002

Biochem. J.

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Results from our molecular-modelling and site-directed-mutagenesis studies of prostaglandin I # synthase (PGIS) have suggested that the large PGIS cytoplasmic domain is anchored to the endoplasmic reticulum (ER) membrane by the N-terminal segment in a way that orients the substrate access channel opening to face the membrane. To test this hypothesis we have explored the accessibility of the PGIS substrate channel opening to site-specific antibodies. The working three-dimensional PGIS model constructed by protein homology modelling was used to predict surface portions near the substrate access channel opening. Two peptides corresponding to the surface immediately near the opening [residues 66-75 (P66-75) and 95-116 (P95-116)], and two other peptides corresponding to the surface about 10-20 A H (1 A H l 0.1 nm) away from the opening [residues 366-382 (P366-382) and 472-482 (P472-482)] were used to prepare sitespecific antibodies. All four antipeptide antibodies specifically recognized the synthetic segments of human PGIS and recombinant PGIS, as shown by binding assays and Western-blot analysis. The site-specific antibodies were used to probe the

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Section: Discussionmentioning

confidence: 99%

Substrate access channel topology in membrane-bound prostacyclin synthase

Deng¹,

Huang²,

So³

et al. 2002

Biochem. J.

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“…Secondly, the residue Phe87, a residue involved in a rotational movement when the substrate is bound, is already almost completely rotated in the substrate-free form of A2 mutant, as in the substrate-bound WT structure ( Figure 3A). Since it has been proposed that the spin equilibrium in P450 BM3 is affected by Phe87, this is an important change since it facilitates both the access of substrates to the active site of the protein and the water displacement [39,40]. Moreover, some residue side chains such as Phe261 and His266 that undergo major changes upon substrate binding, are already in the substrate-bound conformation.…”

Section: Crystal Structures Of the Heme Domain Of A2mentioning

confidence: 99%

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

Nardo

Dell'Angelo

Catucci

et al. 2016

Archives of Biochemistry and Biophysics

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This paper reports the structure of the double mutant Asp251Gly/Gln307His (here called A2), generated by random mutagenesis, is able to produce 4'-hydroxydiclofenac, 2-hydroxyibuprofen and 4-hydroxytolbutamide from diclofenac, ibuprofen and tolbutamide, respectively. Here, we report the crystal structure of the heme domain of the mutant in the substrate-free form and in complex with the substrate N-palmitoylglycine, together with its impact on thermal stability, reduction potential and electron transfer.The substrate-free structure adopts a conformation more similar to the closed one found in the substrate-bound wild type enzyme, but with a higher degree of disorder in the region of the G-helix and F-G loop, part of the substrate access channel. This is due to the mutation Asp251Gly that breaks the salt bridge between Aps251 on Ihelix and Lys224 on G-helix, allowing the G-helix to move away from I-helix and conferring a higher degree of flexibility to this element. This subtle structural change is accompanied by long-range structural rearrangements of the active site with the rotation of Phe87 and a reorganization of catalytically important water molecules.Differential scanning calorimetry shows a population destabilized by 2.2°C compared to the wild type protein, probably reflecting the increased flexibility of part of the protein compared to WT. Moreover, a shift of the heme reduction potential by 50 mV toward positive values, a 2-folds higher first electron transfer rate in the absence of oxygen and a 4-folds higher NADPH consumption rate in the presence of oxygen as the only electron acceptors, when compared to wild type protein.The data demonstrate that a single mutation far away from the active site is able to trigger an increase in protein flexibility in a key region of the substrate access channel, shifting the conformational equilibrium toward the closed form of the protein that is ready to accept electrons and enter the P450 catalytic cycle as soon as a substrate is accepted.

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“…The X-ray structure of the P-450 domain has recently been determined [12], providing a long-awaited atomic model for the class II P-450s.…”

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confidence: 99%

Structural and enzymological analysis of the interaction of isolated domains of cytochrome P‐450 BM3

et al. 1994

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Structural and enzymological analysis of the interaction of isolated domains of cytochrome P-450 BM3 AhstractThe interactions of the individually expressed haem-and flavin-containing domains of cytochrome P-450 BM3 have been analysed by enzymological and spectroscopic techniques. Electron transfer between the isolated domains occurs at a much lower rate than that occurring in the intact flavocytochrome. CD spectroscopic studies indicate that the linkage of the domains in intact P-450 BM3 creates haem and amino acid environments suitable for efficient electron transfer from its flavin domain.Key w&r: ~t~~ro~~ P-450 BM3; Domain inter~t~o~~ Circular dichroism; Electron transfer; Raem environmentThe cytochrome P-450 BM3 system from Bacillus megaterium provides a unique bacterial model for mammalian P-450 systems, such as those involved in drug and xenobiotic metabolism [l-3]. It is the only characterised prokaryotic class II P-450 utilising an FADIFMN containing NADPH-P-450 reductase as an electron transfer partner [4]. Other prokaryotic (class I) systems operate a three protein-~om~nent electron transfer chain with electrons flowing from NAD(P)H onto an FADcontaining redoxin reductase, through an iron-sulphur redoxin and onto the P-450 (e.g. [5]). In addition, P-450 BM3 has its redox partners fused in a single polypeptide chain, with the haem-containing P-450 'domain' being N-terminal and the flavincontaining reductase C-terminal [4,6]. The only structurally analogous P-450 yet characterised is the cell signalling enzyme nitric oxide (NO) synthase [71-Like all proka~oti& P-45&, P-450 BM3 is a soluble enzyme. This has enabled relatively simple p~~~a~on of the ~l~p~de [4]. The gene has been cloned and expressed in E, co& [S,9], as have the sub-genes encoding *Corresponding author. Fax: (44) (41) 330 4620.

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Crystal Structure of Hemoprotein Domain of P450BM-3, a Prototype for Microsomal P450's

Cited by 912 publications

References 33 publications

Substrate access channel topology in membrane-bound prostacyclin synthase

Substrate access channel topology in membrane-bound prostacyclin synthase

Subtle structural changes in the Asp251Gly/Gln307His P450 BM3 mutant responsible for new activity toward diclofenac, tolbutamide and ibuprofen

Structural and enzymological analysis of the interaction of isolated domains of cytochrome P‐450 BM3

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