Inhibitor/fatty acid interactions with cytochrome P‐450 BM3

Macdonald, Iain D. G.; Smith, W. Ewen; Munro, Andrew W.

doi:10.1016/0014-5793(96)01095-2

Cited by 25 publications

(24 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The values of K D of the complexes of P450-SS9 with the fatty acids with the chain length of 12-18 carbon atoms falls into the range of 1 – 25 μM (Fig. 3, Table 1), which is typical of known fatty acid hydroxylases, such as P450BM-3 (41, 42), P450foxy (43) or P450 4A1 (44, 45). As illustrated in Fig.…”

Section: Resultsmentioning

confidence: 90%

Cytochrome P450 fromPhotobacterium profundumSS9, a Piezophilic Bacterium, Exhibits a Tightened Control of Water Access to the Active Site

Sineva

Davydov

2010

Biochemistry

View full text Add to dashboard Cite

We report cloning, expression in E. coli, and purification of cytochrome P450 from a deep-sea bacterium Photobacterium profundum strain SS9 (P450-SS9). The enzyme, which is predominately high-spin (86%) in the absence of any added ligand, binds fatty acids and their derivatives and exhibits the highest affinity for myristic acid. Binding of the majority of saturated fatty acids displaces the spin equilibrium further towards the high-spin state, whereas the interactions with unsaturated fatty acids and their derivatives (arachidonoyl glycine) have the opposite effect. Pressure perturbation studies showed that increasing pressure fails to displace the spin equilibrium completely to the low-spin state in the ligand-free P450-SS9 or in the complexes with either myristic acid or arachidonoyl glycine. Stabilization of high-spin P450-SS9 signifies a pressure-induced transition to a state with reduced accessibility of the active site. This transition, which is apparently associated with substantial hydration of the protein, is characterized by the reaction volume change (ΔV) around −100 -−200 mL/mol and P ½ of 300-800 bar, which is close to the pressure of habitation of P. profundum. The transition to a state with confined water accessibility is hypothesized to represent a common feature of cytochromes P450 that serves to coordinate heme pocket hydration with ligand binding and the redox state. Displacement of the conformational equilibrium towards the "closed" state in P450-SS9 (even ligand-free) may have evolved to allow the protein to adapt to enhanced protein hydration at high hydrostatic pressures.The interactions of proteins with solvent and, in particular, changes in protein hydration due to intermolecular interactions and ligand-induced conformational rearrangements are known to be of fundamental functional importance (1-5). The fact that protein interactions with solvent are highly sensitive to hydrostatic pressure (6-10) warrants the use of a pressureperturbation approach to study the dynamics of protein bound water and its role in proteinligand, protein-protein, and protein-DNA interactions (1,(11)(12)(13). This approach is especially valuable for studies of heme proteins, where the presence of the heme chromophore allows the use of various spectroscopic techniques for monitoring the transitions between different states of the enzyme. On the other hand, the sensitivity of protein hydration to hydrostatic pressure creates a challenge for structural adaptation of proteins from piezophilic organisms, such as deep-sea bacteria, in order to function at extreme pressures. The role of this adaptation is especially important in the case of proteins whose functional mechanisms involve hydration-dehydration dynamics. The structural and functional comparison of proteins from deep-sea organisms with their homologs from non-piezophilic species may help to elucidate the functional role of protein hydration and provide important information † This research was supported by NIH grant GM54995 on the dynamics of protei...

show abstract

Section: Resultsmentioning

confidence: 90%

Cytochrome P450 fromPhotobacterium profundumSS9, a Piezophilic Bacterium, Exhibits a Tightened Control of Water Access to the Active Site

Sineva

Davydov

2010

Biochemistry

View full text Add to dashboard Cite

show abstract

“…This makes it possible to probe the structure of the active pocket in situ in aqueous solution in physiologically useful concentrations. Previous studies have used surface‐enhanced resonance Raman scattering to probe the Q‐band structure4, 5 and resonance Raman scattering to probe the Soret band 6…”

Section: Introductionmentioning

confidence: 99%

Resonance Raman scattering of cytochrome P450 BM3 and effect of imidazole inhibitors

2003

View full text Add to dashboard Cite

Resonance Raman scattering from cytochrome P450 BM3 is obtained with a Raman microprobe using 406-nm excitation with an accumulation time of a few seconds. The small sample size and rapid measurement time make the routine characterization of P450 systems by resonance Raman spectroscopy easier. Addition of imidazole and imidazole derivatives as inhibitors causes the appearance of additional peaks due to vinyl modes, increases the relative intensity of symmetric modes that would be A(1g) in D(4h) symmetry, and causes a large drop in the intensity of nu(11). This information indicates that the ligation of imidazoles to the heme iron causes the alignment of the vinyl modes with the plane of the heme ring and reduces the out of plane distortion of the ring. The effect of both inhibitors is similar but there is a subtle difference in the extent of the reduction in the intensity of nu(11), which suggests that steric effects within the pocket are having some effect.

show abstract

“…The shift to Schs has been observed with other P450s and is caused by partial water displacement upon surface adsorption, with no obvious disruption to the active site topology (2). However, there is also a contribution from six coordinate low spin …”

mentioning

confidence: 56%

“…SERRS has already been successfully applied to haemoproteins (I), enabling interpretation of oxidation, coordination and the environment around the haem chromophore of proteins at concentrations far lower than that detectable by resonance Raman scattering (2).…”

mentioning

confidence: 99%

A Single Amino Acid (Glu146) governs the substrate specificity of human catecholamine sulfotransferase SULT1A3

Dajani

Hood

Coughtrie

1999

Biochemical Society Transactions

View full text Add to dashboard Cite

Sulfation is an important metabolic event in the homeostasis of numerous biologically potent endogenous chemicals such as catecholamines, steroids and iodothyronines, as well as in the detoxication of xenobiotics. SULTlA3 (or M-PST) shares 93% sequence identity with SULTlAl (or P-PST) but has very different substrate preferencehpecificity. SULTlA3 prefers catecholamines (e.g. dopamine and tyramine), SULTlAl prefers phenols (e.g. 4nitrophenol). This provides an ideal system to study which amino acids are important in substrate specificity. Amino acid sequence alignment analysis of sulfotransferases and examination of the recently determined crystal structure of mouse estrogen sulfotransferase, reveals important putative substrate binding domains containing amino acids that are potentially crucial in determining substrate specificity in SULTlA3 and SULTlAl. Two charged amino acids in SULTlA3, His143 and Glu146, were mutated to the corresponding amino acids in SULTlAl. Two single mutants and one double mutant, combining both changes, were generated as follows : lA3H143Y; 1A3E146A; lA3H143YE146A. Kinetic analysis using the three mutants and wild types SULTlA3 and SULTlAl, revealed that the E146A mutation resulted in a 50 fold increase in the Km towards dopamine and a similar Km towards 4-nitrophenol as that of SULTlAl, suggesting that amino acid 146 is essential in the interaction with substrates. The mutation H143Y did not have a marked effect on the enzymes affinity towards dopamine and 4-nitrophenol. Thus we have determined that Glu146 is a major contributor towards SULTlAl's ability to sulfate catechols.

show abstract

Inhibitor/fatty acid interactions with cytochrome P‐450 BM3

Cited by 25 publications

References 25 publications

Cytochrome P450 fromPhotobacterium profundumSS9, a Piezophilic Bacterium, Exhibits a Tightened Control of Water Access to the Active Site

Cytochrome P450 fromPhotobacterium profundumSS9, a Piezophilic Bacterium, Exhibits a Tightened Control of Water Access to the Active Site

Resonance Raman scattering of cytochrome P450 BM3 and effect of imidazole inhibitors

A Single Amino Acid (Glu146) governs the substrate specificity of human catecholamine sulfotransferase SULT1A3

Contact Info

Product

Resources

About