Stephen K. Chapman

P450 BM3: the very model of a modern flavocytochrome

Flavocytochrome P450 BM3 is a bacterial P450 system in which a fatty acid hydroxylase P450 is fused to a mammalian-like diflavin NADPH-P450 reductase in a single polypeptide. The enzyme is soluble (unlike mammalian P450 redox systems) and its fusion arrangement affords it the highest catalytic activity of any P450 mono-oxygenase. This article discusses the fundamental properties of P450 BM3 and how progress with this model P450 has affected our comprehension of P450 systems in general.

Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase

Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-Å resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate l-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the l-stereospecificity. A second, possibly allosteric, l-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of l-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.

Atomic structure of Mycobacterium tuberculosis CYP121 to 1.06 A reveals novel features of cytochrome P450.

The first structure of a P450 to an atomic resolution of 1.06 Å has been solved for CYP121 fromMycobacterium tuberculosis. A comparison with P450 EryF (CYP107A1) reveals a remarkable overall similarity in fold with major differences residing in active site structural elements. The high resolution obtained allows visualization of several unusual aspects. The heme cofactor is bound in two distinct conformations while being notably kinked in one pyrrole group due to close interaction with the proline residue (Pro346) immediately following the heme iron-ligating cysteine (Cys345). The active site is remarkably rigid in comparison with the remainder of the structure, notwithstanding the large cavity volume of 1350 Å3. The region immediately surrounding the distal water ligand is remarkable in several aspects. Unlike other bacterial P450s, the I helix shows no deformation, similar to mammalian CYP2C5. In addition, the positively charged Arg386 is located immediately above the heme plane, dominating the local structure. Putative proton relay pathways from protein surface to heme (converging at Ser279) are identified. Most interestingly, the electron density indicates weak binding of a dioxygen molecule to the P450. This structure provides a basis for rational design of putative antimycobacterial agents.

Rational re-design of the substrate binding site of flavocytochrome P450 BM3

Bacillus megaterium P450 BM3 is a fatty acid hydroxylase with selectivity for long chain substrates (C12–C20). Binding or activity with substrates of chain length 13‐fold with butyrate, while the L75T/L181K double mutant has k cat/K M increased >15‐fold with hexanoate and binding (K d) improved >28‐fold for butyrate. Removing the arginine 47/lysine 51 carboxylate binding motif at the mouth of the active site disfavours binding of all fatty acids, indicating its importance in the initial recognition of substrates.

Expression, purification and spectroscopic characterization of the cytochrome p450 cyp121 from mycobacterium tuberculosis

The CYP121 gene from the pathogenic bacterium Mycobacterium tuberculosis has been cloned and expressed in Escherichia coli, and the protein purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The CYP121 gene encodes a cytochrome P450 enzyme (CYP121) that displays typical electronic absorption features for a member of this superfamily of hemoproteins (major Soret absorption band at 416.5 nm with alpha and beta bands at 565 and 538 nm, respectively, in the oxidized form) and which binds carbon monoxide to give the characteristic Soret band shift to 448 nm. Resonance Raman, EPR and MCD spectra show the protein to be predominantly low-spin and to have a typical cysteinate- and water-ligated b-type heme iron. CD spectra in the far UV region describe a mainly alpha helical conformation, but the visible CD spectrum shows a band of positive sign in the Soret region, distinct from spectra for other P450s recognized thus far. CYP121 binds very tightly to a range of azole antifungal drugs (e.g. clotrimazole, miconazole), suggesting that it may represent a novel target for these antibiotics in the M. tuberculosis pathogen.

Octaheme tetrathionate reductase is a respiratory enzyme with novel heme ligation

We have isolated a soluble cytochrome from Shewanella oneidensis that contains eight covalently attached heme groups and determined its crystal structure. One of these hemes exhibits novel ligation of the iron atom by the ε-amino group of a lysine residue, despite its attachment via a typical CXXCH motif. This heme is most likely the active site for tetrathionate reduction, a reaction catalyzed efficiently by this enzyme.

Protein engineering of cytochromes P-450

The cytochromes P-450 are an immensely important superfamily of heme-containing enzymes. They catalyze the monooxygenation of an enormous range of substrates. In bacteria, cytochromes P-450 are known to catalyze the hydroxylation of environmentally significant substrates such as camphor, phenolic compounds and many herbicides. In eukaryotes, these enzymes perform key roles in the synthesis and interconversion of steroids, while in mammals hepatic cytochromes P-450 are vital for the detoxification of many drugs. As such, the cytochromes P-450 are of considerable interest in medicine and biotechnology and are obvious targets for protein engineering. The purpose of this article is to illustrate the ways in which protein engineering has been used to investigate and modify the properties of cytochromes P-450. Illustrative examples include: the manipulation of substrate selectivity and regiospecificity, the alteration of membrane binding properties, and probing the route of electron transfer.

Exploring the mechanism of tryptophan 2,3-dioxygenase.

The haem proteins TDO (tryptophan 2,3-dioxygenase) and IDO (indoleamine 2,3-dioxygenase) are specific and powerful oxidation catalysts that insert one molecule of dioxygen into L-tryptophan in the first and rate-limiting step in the kynurenine pathway. Recent crystallographic and biochemical analyses of TDO and IDO have greatly aided our understanding of the mechanisms employed by these enzymes in the binding and activation of dioxygen and tryptophan. In the present paper, we briefly discuss the function, structure and possible catalytic mechanism of these enzymes.

Reassessment of the Reaction Mechanism in the Heme Dioxygenases

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme enzymes that catalyze the O(2)-dependent oxidation of L-tryptophan to N-formyl-kynurenine. Previous proposals for the mechanism of this reaction have suggested that deprotonation of the indole NH group, either by an active-site base or by oxygen bound to the heme iron, as the initial step. In this work, we have examined the activity of 1-Me-L-Trp with three different heme dioxygenases and their site-directed variants. We find, in contrast to previous work, that 1-Me-L-Trp is a substrate for the heme dioxygenase enzymes. These observations suggest that deprotonation of the indole N(1) is not essential for catalysis, and an alternative reaction mechanism, based on the known chemistry of indoles, is presented.

Identification and characterization of a novel cytochrome c(3) from Shewanella frigidimarina that is involved in Fe(III) respiration.

Shewanella frigidimarina NCIMB400 is a non-fermenting, facultative anaerobe from the gamma group of proteobacteria. When grown anaerobically this organism produces a wide variety of periplasmic c-type cytochromes, mostly of unknown function. We have purified a small, acidic, low-potential tetrahaem cytochrome with similarities to the cytochromes c(3) from sulphate-reducing bacteria. The N-terminal sequence was used to design PCR primers and the cctA gene encoding cytochrome c(3) was isolated and sequenced. The EPR spectrum of purified cytochrome c(3) indicates that all four haem irons are ligated by two histidine residues, a conclusion supported by the presence of eight histidine residues in the polypeptide sequence, each of which is conserved in a related cytochrome c(3) and in the cytochrome domains of flavocytochromes c(3). All four haems exhibit low midpoint redox potentials that range from -207 to -58 mV at pH 7; these values are not significantly influenced by pH changes. Shewanella cytochrome c(3) consists of a mere 86 amino acid residues with a predicted molecular mass of 11780 Da, including the four attached haem groups. This corresponds closely to the value of 11778 Da estimated by electrospray MS. To examine the function of this novel cytochrome c(3) we constructed a null mutant by gene disruption. S. frigidimarina lacking cytochrome c(3) grows well aerobically and its growth rate under anaerobiosis with a variety of electron acceptors is indistinguishable from that of the wild-type parent strain, except that respiration with Fe(III) as sole acceptor is severely, although not completely, impaired.