Max J. Cryle

Structural insights from a P450 Carrier Protein complex reveal how specificity is achieved in the P450(BioI) ACP complex.

Cytochrome P450BioI (CYP107H1) from the biotin operon of Bacillus subtilis forms a seven-carbon diacid through a multistep oxidative cleavage of a fatty acid linked to acyl carrier protein (ACP). Crystal structures of P450BioI in complex with three different length fatty acyl-ACP (Escherichia coli) ligands show that P450BioI binds the fatty acid such as to force the carbon chain into a U-shape above the active site heme. This positions the C7 and C8 carbons for oxidation, with a large additional cavity extending beyond the heme to accommodate the methyl termini of fatty acids beyond the site of cleavage. The structures explain the experimentally observed lack of stereo- and regiospecificity in the hydroxylation and cleavage of free fatty acids. The P450BioI-ACP complexes represent the only structurally characterized P450-carrier protein complexes to date, which has allowed the generation of a model of the interaction of the vancomycin biosynthetic P450 OxyB with its proposed carrier protein bound substrate.

Reactions Catalyzed by Bacterial Cytochromes P450

The cytochromes P450 are a large family of oxidative haemoproteins that are responsible for a wide variety of oxidative transformations in a variety of organisms. This review focuses upon the reactions catalyzed specifically by bacterial enzymes, which includes aliphatic hydroxylation, alkene epoxidation, aromatic hydroxylation, oxidative phenolic coupling, heteroatom oxidation and dealkylation, and multiple oxidations including C-C bond cleavage. The potential for the practical application of the oxidizing power of these enzymes is briefly discussed.

Arachidonic acid potentiates exocytosis and allows neuronal SNARE complex to interact with Munc18a

Neuronal communication relies on the fusion of neurotransmitter‐containing vesicles with the neuronal plasma membrane. Recent genetic studies have highlighted the critical role played by polyunsaturated fatty acids in neurotransmission, however, there is little information available about which fatty acids act on exocytosis and, more importantly, by what mechanism. We have used permeabilized chromaffin cells to screen various fatty acids of the n‐3 and n‐6 series for their acute effects on exocytosis. We have demonstrated that an n‐6 series polyunsaturated fatty acid, arachidonic acid, potentiates secretion from intact neurosecretory cells regardless of the secretagogue used. We have shown that arachidonic acid dose dependently increases soluble NSF attachment protein receptor complex formation in chromaffin cells and bovine cortical brain extracts and that a non‐hydrolysable analogue of arachidonic acid causes a similar increase in SNARE complex formation. This prompted us to examine the effect of arachidonic acid on SNARE protein interactions with Munc18a, a protein known to prevent Syntaxin1a engagement into the SNARE complex in vitro. In the presence of arachidonic acid, we show that Munc18a can interact with the neuronal SNARE complex in a dose‐dependent manner. We further demonstrate that arachidonic acid directly interacts with Syntaxin1a.

X-domain of peptide synthetases recruits oxygenases crucial for glycopeptide biosynthesis

Non-ribosomal peptide synthetase (NRPS) mega-enzyme complexes are modular assembly lines that are involved in the biosynthesis of numerous peptide metabolites independently of the ribosome. The multiple interactions between catalytic domains within the NRPS machinery are further complemented by additional interactions with external enzymes, particularly focused on the final peptide maturation process. An important class of NRPS metabolites that require extensive external modification of the NRPS-bound peptide are the glycopeptide antibiotics (GPAs), which include vancomycin and teicoplanin. These clinically relevant peptide antibiotics undergo cytochrome P450-catalysed oxidative crosslinking of aromatic side chains to achieve their final, active conformation. However, the mechanism underlying the recruitment of the cytochrome P450 oxygenases to the NRPS-bound peptide was previously unknown. Here we show, through in vitro studies, that the X-domain, a conserved domain of unknown function present in the final module of all GPA NRPS machineries, is responsible for the recruitment of oxygenases to the NRPS-bound peptide to perform the essential side-chain crosslinking. X-ray crystallography shows that the X-domain is structurally related to condensation domains, but that its amino acid substitutions render it catalytically inactive. We found that the X-domain recruits cytochrome P450 oxygenases to the NRPS and determined the interface by solving the structure of a P450–X-domain complex. Additionally, we demonstrated that the modification of peptide precursors by oxygenases in vitro—in particular the installation of the second crosslink in GPA biosynthesis—occurs only in the presence of the X-domain. Our results indicate that the presentation of peptidyl carrier protein (PCP)-bound substrates for oxidation in GPA biosynthesis requires the presence of the NRPS X-domain to ensure conversion of the precursor peptide into a mature aglycone, and that the carrier protein domain alone is not always sufficient to generate a competent substrate for external cytochrome P450 oxygenases.

The Structure of a Transient Complex of a Nonribosomal Peptide Synthetase and a Cytochrome P450 Monooxygenase

Studying the interplay between nonribosomal peptide synthetases (NRPS), a major source of secondary metabolites, and crucial external modifying enzymes is a challenging task since the interactions involved are often transient in nature. By applying a range of synthetic inhibitor-type compounds, a stabilized complex appropriate for structural analysis was generated for such a tailoring enzyme and an NRPS domain. The complex studied comprises an NRPS peptidyl carrier protein (PCP) domain bound to the Cytochrome P450 enzyme that is crucial for the provision of β-hydroxylated amino acid precursors in the biosynthesis of the cyclic depsipeptide skyllamycin. The structure reveals that complex formation is governed by hydrophobic interactions, the presence of which can be controlled through minor alterations in PCP structure that enable selectivity amongst multiple highly similar PCP domains.

Structural characterization of oxyd, a cytochrome p450 involved in β-hydroxytyrosine formation in vancomycin biosynthesis

The cytochrome P450 OxyD from the balhimycin glycopeptide antibiotic biosynthetic operon of Amycolatopsis mediterranei is involved in the biosynthesis of the modified amino acid β-R-hydroxytyrosine, an essential precursor for biosynthesis of the vancomycin-type aglycone. OxyD binds the substrate tyrosine not free in solution, but rather covalently linked to the carrier protein (CP) domain of the non-ribosomal peptide synthase BpsD, exhibiting micromolar binding affinity to a tyrosine-loaded carrier protein construct. The crystal structure of OxyD was determined to 2.1-Å resolution, revealing a potential binding site for the carrier protein-bound substrate in a different orientation to that seen with the acyl carrier protein-bound P450BioI (Cryle, M. J., and Schlichting, I. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 15696–15701). A series of residues were identified across known aminoacyl-CP-oxidizing P450s that are highly conserved and cluster in the active site or potential CP binding site of OxyD. These residues appear to be characteristic for aminoacyl-CP-oxidizing P450s, allowing sequence based identification of P450 function for this subgroup of P450s that play vital roles in the biosyntheses of many important natural products in addition to the vancomycin-type antibiotics. The ability to analyze such P450 function based upon sequence data alone should prove an important tool in the analysis and identification of new medicinally relevant biomolecules.

Femtosecond free-electron laser x-ray diffraction data sets for algorithm development.

We describe femtosecond X-ray diffraction data sets of viruses and nanoparticles collected at the Linac Coherent Light Source. The data establish the first large benchmark data sets for coherent diffraction methods freely available to the public, to bolster the development of algorithms that are essential for developing this novel approach as a useful imaging technique. Applications are 2D reconstructions, orientation classification and finally 3D imaging by assembling 2D patterns into a 3D diffraction volume.

The archaeal cofactor F0 is a light-harvesting antenna chromophore in eukaryotes

Archae possess unique biochemical systems quite distinct from the pathways present in eukaryotes and eubacteria. 7,8-Dimethyl-8-hydroxy-5deazaflavin (F0) and F420 are unique deazaflavin-containing coenzyme and methanogenic signature molecules, essential for a variety of biochemical transformations associated with methane biosynthesis and light-dependent DNA repair. The deazaflavin cofactor system functions during methane biosynthesis as a low-potential hydrid shuttle F420/F420H2. In DNA photolyase repair proteins, the deazaflavin cofactor is in the deprotonated state active as a light-collecting energy transfer pigment. As such, it converts blue sunlight into energy used by the proteins to drive an essential repair process. Analysis of a eukaryotic (6-4) DNA photolyase from Drosophila melanogaster revealed a binding pocket, which tightly binds F0. Residues in the pocket activate the cofactor by deprotonation so that light absorption and energy transfer are switched on. The crystal structure of F0 in complex with the D. melanogaster protein shows the atomic details of F0 binding and activation, allowing characterization of the residues involved in F0 activation. The results show that the F0/F420 coenzyme system, so far believed to be strictly limited to the archael kingdom of life, is far more widespread than anticipated. Analysis of a D. melanogaster extract and of a DNA photolyase from the primitive eukaryote Ostreococcus tauri provided direct proof for the presence of the F0 cofactor also in higher eukaryotes.

Cytochrome p450sky interacts directly with the nonribosomal Peptide synthetase to generate three amino Acid precursors in skyllamycin biosynthesis.

The generation of modified amino acid precursors for incorporation in nonribosomal peptide synthesis (NRPS) plays a crucial, if often understated, role in the generation of peptide natural products. The biosynthesis of the cyclic depsipeptide skyllamycin requires three β-hydroxylated amino acid precursors, with in vivo gene inactivation experiments implicating cytochrome P450sky (CYP163B3) in the hydroxylation of these amino acids. Here, we demonstrate the in vitro oxidation of l-amino acid substrates bound to peptidyl carrier protein (PCP) domains 5, 7, and 11 of the skyllamycin nonribosomal synthetase by P450sky. Selectivity for these domains over other PCP domains could be demonstrated, with hydroxylation selective for l-amino acids and stereospecific in nature resulting in the (2S,3S)-configuration. The oxidation of amino acids or small molecule substrate analogues was not supported, demonstrating the necessity of the carrier protein in P450sky-catalyzed hydroxylation. The binding of aminoacyl-PCP substrates to P450sky was detected for the catalytically active PCP7 but not for the catalytically inactive PCP10, indicating carrier protein-mediated selectivity in P450sky substrate binding. X-ray crystal structures of P450sky reveal a 3D-structure with a highly open active site, the size of which is dictated by the carrier protein bound nature of the substrate. P450sky is the first P450 demonstrated to not only interact directly with PCP-bound amino acids within the peptide-forming NRPS but also to do so with three different PCP domains in a specific fashion. This represents an expansion of the complexity and scope of NRPS-mediated peptide synthesis, with the generation of hydroxylated amino acid precursors occurring through the interaction of P450 enzymes following, rather than prior to, the selection of amino acids by NRPS-adenylation domains.

Calibration of the channel that determines the ω-Hydroxylation regiospecificity of cytochrome P4504A1: catalytic oxidation of 12-HALODOdecanoic acids

The fatty acid ω-hydroxylation regiospecificity of CYP4 enzymes may result from presentation of the terminal carbon to the oxidizing species via a narrow channel that restricts access to the other carbon atoms. To test this hypothesis, the oxidation of 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids by recombinant CYP4A1 has been examined. Although all three 12-halododecanoic acids bind to CYP4A1 with similar dissociation constants, the 12-chloro and 12-bromo fatty acids are oxidized to 12-hydroxydodecanoic acid and 12-oxododecanoic acid, whereas the 12-iodo analogue is very poorly oxidized. Incubations in H2182O show that the 12-hydroxydodecanoic acid oxygen derives from water, whereas that in the aldehyde derives from O2. The alcohol thus arises from oxidation of the halide to an oxohalonium species that is hydrolyzed by water, whereas the aldehyde arises by a conventional carbon hydroxylation-elimination mechanism. No irreversible inactivation of CYP4A1 is observed during 12-halododecanoic acid oxidation. Control experiments show that CYP2E1, which has an ω-1 regiospecificity, primarily oxidizes 12-halododecanoic acids to the ω-aldehyde rather than alcohol product. Incubation of CYP4A1 with 12,12-[2H]2-12-chlorododecanoic acid causes a 2–3-fold increase in halogen versus carbon oxidation. The fact that the order of substrate oxidation (Br > Cl » I) approximates the inverse of the intrinsic oxidizability of the halogen atoms is consistent with presentation of the halide terminus via a channel that accommodates the chloride and bromide but not iodide atoms, which implies an effective channel diameter greater than 3.90 Å but smaller than 4.30 Å.