Martin P. Mayhew

Probing the interactions of putidaredoxin with redox partners in camphor P450 5-monooxygenase by mutagenesis of surface residues.

The role of surface amino acid residues in the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam (CYP101) system was investigated by site-directed mutagenesis. The mutated Pdx genes were expressed inEscherichia coli, and the proteins were purified and studied in vitro. Activity of the complete reconstituted P450cam system was measured, and kinetic parameters were determined. Partial assays were also conducted to determine the effect of the mutations on interactions with each redox partner. Some mutations altered interactions of Pdx with one redox partner but not the other. Other mutations affected interactions with both redox partners, suggesting some overlap in the binding sites on Pdx for putidaredoxin reductase and CYP101. Cysteine 73 of Pdx was identified as important in the interaction of Pdx with putidaredoxin reductase, whereas aspartate 38 serves a critical role in the subunit binding and electron transfer to CYP101.

The crystal structure of chorismate lyase shows a new fold and a tightly retained product.

The enzyme chorismate lyase (CL) catalyzes the removal of pyruvate from chorismate to produce 4‐hydroxy benzoate (4HB) for the ubiquinone pathway. In Escherichia coli, CL is monomeric, with 164 residues. We have determined the structure of the CL product complex by crystallographic heavy‐atom methods and report the structure at 1.4‐Å resolution for a fully active double Cys‐to‐Ser mutant and at 2.0‐Å resolution for the wild‐type. The fold involves a 6‐stranded antiparallel β‐sheet with no spanning helices and novel connectivity. The product is bound internally, adjacent to the sheet, with its polar groups coordinated by two main‐chain amides and by the buried side‐chains of Arg 76 and Glu 155. The 4HB is completely sequestered from solvent in a largely hydrophobic environment behind two helix–turn–helix loops. The extensive product binding that is observed is consistent with biochemical measurements of slow product release and 10‐fold stronger binding of product than substrate. Substrate binding and kinetically rate‐limiting product release apparently require the rearrangement of these active‐site‐covering loops. Implications for the biological function of the high product binding are considered in light of the unique cellular role of 4HB, which is produced by cytoplasmic CL but is used by the membrane‐bound enzyme 4HB octaprenyltransferase. Proteins 2001;44:304–311.

Chorismate lyase: kinetics and engineering for stability.

By removing the enolpyruvyl group from chorismate, chorismate lyase (CL) produces p-hydroxybenzoate (p-HB) for the ubiquinone biosynthetic pathway. We have analyzed CL by several spectroscopic and chemical techniques and measured its kinetic (kcat=1.7 s(-1), K(m)=29 microM) and product inhibition parameters (K(p)=2.1 microM for p-HB). Protein aggregation, a serious problem with wild type CL, proved to be primarily due to the presence of two surface-active cysteines, whose chemical modification or mutation (to serines) gave greatly improved solution behavior and minor effects on enzyme activity. CL is strongly inhibited by its product p-HB; for this reason activity and inhibition measurements were analyzed by both initial rate and progress curve methods. The results are consistent, but in this case where the stable enzyme-product complex rapidly becomes the predominant form of the enzyme, progress curve methods are more efficient. We also report inhibition measurements with several substrate and product analogs that give information on ligand binding interactions of the active site. The biological function of the unusual product retention remains uncertain, but may involve a mechanism of directed delivery to the membrane-bound enzyme that follows CL in the ubiquinone pathway.

Improving the Cytochrome P450 Enzyme System for Electrode-Driven Biocatalysis of Styrene Epoxidation

Cytochrome P450 enzymes catalyze a vast array of oxidative and reductive biotransformations that are potentially useful for industrial and pharmaceutical syntheses. Factors such as cofactor utilization and slow reaction rates for nonnatural substrates limit their large‐scale usefulness. This paper reports several improvements that make the cytochrome P450cam enzyme system more practical for the epoxidation of styrene. NADH coupling was increased from 14 to 54 mol %, and product turnover rate was increased from 8 to 70 min−1 by introducing the Y96F mutation to P450cam. Styrene and styrene oxide mass balance determinations showed different product profiles at low and high styrene conversion levels. For styrene conversion less than about 25 mol %, the stoichiometry between styrene consumption and styrene oxide formation was 1:1. At high styrene conversion, a second doubly oxidized product, α‐hydroxyacetophenone, was formed. This was also the exclusive product when Y96F P450cam acted on racemic, commercially available styrene oxide. The α‐hydroxyacetophenone product was suppressed in reactions where styrene was present at saturating concentrations. Finally, styrene epoxidation was carried out in an electroenzymatic reactor. In this scheme, the costly NADH cofactor and one of the three proteins (putidaredoxin reductase) are eliminated from the Y96F P450cam enzyme system.

Redox control of the P450cam catalytic cycle: effects of Y96F active site mutation and binding of a non-natural substrate

Spectroelectrochemistry measurements are used to demonstrate that active site mutation and binding of an non-natural substrate to P450cam (CYP101) reduces the shift in the redox potential caused by substrate-binding, and thereby results in slower catalytic turnover rate relative to wild-type enzyme with the natural camphor substrate.

Benzocycloarene hydroxylation by P450 biocatalysis

Experimental and theoretical studies of the hydroxylation of a family of benzocycloarene compounds [benzocyclobutene, benzocyclopentene (indan), benzocyclohexene (tetralin), and benzocycloheptene] by wild type and Y96F mutant P450cam were performed in order to understand the factors affecting product distribution, catalytic rate and cofactor utilization. The products of all reactions except that of benzocycloheptene were regiospecifically hydroxylated in the 1-position. Reaction energetics predominated over active site steric constraints in this case so that quantum mechanical calculations (B3LYP/6-31G*) comparing the energetics of all possible radical intermediates successfully predicted hydroxylation at the 1- and 3-positions of benzocycloheptene, and at the 1-position for the other three compounds. However, the fact that the ratio of 1-alcohol to 3-alcohol changes significantly between wild type and Y96F mutant P450cam indicates that active site geometry and composition also play a significant role in determining BCA7 product regiospecificity. The indan and tetralin reaction products were stereoselective for the R enantiomer (88 and 94%, respectively). Steric constraints of the active site were confirmed by molecular dynamics calculations (locally enhanced sampling dynamics) to control enantiomer distribution for tetralin hydroxylation. NADH coupling, binding affinity, and product turnover rates were dramatically higher for Y96F P450cam, showing that the removal of the active site hydroxyl group on tyrosine makes the enzyme better suited for oxidation of these hydrophobic compounds. NADH coupling, binding affinity and product turnover rate for each enzyme generally increased with arene ring size. For both enzymes, NADH coupling and product turnover rates were correlated with the extent of high-spin shift upon substrate binding as determined by the shift in Soret absorption bands at 417 and 391 nm.

New degenerate metal-oxide electrodes for nearly reversible direct electron transfer to the redox proteins

Abstract Heavily doped cadmium tin oxide (CTO) film electrodes were developed for fast electron exchange with redox proteins. The metal oxide films showed nearly reversible electron transfer for the [2Fe–2S] proteins spinach ferredoxin (Sp fd) and putidaredoxin (Pdx), and the well-studied heme protein horse heart cytochrome c. These represent a family of proteins that are of comparable size, but vary significantly in overall charge, formal redox potential, and type of metal center. The unmediated electron exchange was achieved through variation of metal oxide film synthesis parameters that led to an increase of the charge carrier concentration up to the levels typical for degenerate semiconductors. In addition, the flat band potential of the films was shifted close to or more positive of the formal redox potentials of proteins such that the semiconductor electrodes would be utilized in an accumulation mode. The rates and sustainability of electron transfer for the two ferredoxins obtained on these cadmium tin oxide electrodes are as high or higher than previously reported.

Crystallization and phasing of alanine dehydrogenase from Archaeoglobus fulgidus

Alanine dehydrogenase (AlaDH) from the hyperthermophilic archaeon Archaeoglobus fulgidus is a dimer of 35 kDa chains. The archaeal enzyme appears to represent a new class of AlaDH that is not homologous to bacterial AlaDH enzymes, but has close evolutionary links to the broad ornithine cyclodeaminase/micro-crystallin family, which includes human thyroid hormone binding protein, which has 30% sequence identity to the A. fulgidus gene. The enzyme has been cloned, shown to catalyze the NAD-dependent interconversion of alanine and pyruvate and crystallized in several forms. Although the purified protein crystallized readily under many conditions, most of the crystals diffracted weakly or not at all. One polymorph growing in space group P2(1)2(1)2(1) has non-crystallographic symmetry that becomes crystallographic, changing the space group to P2(1)2(1)2, upon binding iridium or samarium. Before and after derivatization, these crystals diffracted to 2.5 A using synchrotron radiation. Multiwavelength diffraction data were collected from the non-isomorphous iridium derivative, enabling structure determination.

Structure of C73G putidaredoxin from Pseudomonas putida

The structure of the C73G mutant of putidaredoxin (Pdx), the Fe(2)S(2) ferredoxin that supplies electrons to cytochrome CYP101 (p450cam) for camphor oxidation, is reported at 1.9 A resolution in a C2 crystal form. The structure was solved by single-wavelength iron anomalous diffraction, which yielded electron density above the 2sigma level for over 97% of the non-H atoms in the protein. The final structure with R = 0.19 and R(free) = 0.21 has been deposited in the Protein Data Bank with accession code 1r7s. The C2 crystal contains three Pdx molecules in the asymmetric unit, giving three independent models of the protein that are very similar (r.m.s.d. < 0.3 A for the 106 C(alpha) atoms). The unusually high solvent fraction of 80% results in comparatively few crystal-packing artifacts. The structure is briefly compared with the recently reported crystal structures of the C73S and C73S/C85S mutants. In general, the eight independent molecules in the three crystal structures (three in C73G, three in C73S and two in C73S/C85S) are much more similar to each other than to the previously reported NMR structure of wild-type Pdx in solution. The present findings show a unanimous structure in some regions crucial for electron-transfer interactions, including the cluster-binding loop 39-48 and the cytochrome-interaction region of Asp38 and Trp106. In addition, the Cys45 amide group donates a hydrogen bond to cluster sulfur S1, with Ala46 adopting an Lalpha conformation, in all three molecules in the crystal.

Structural alterations of the heme environment of cytochrome P450cam and the Y96F mutant as deduced by resonance Raman spectroscopy

Resonance Raman spectroscopy at 2.5cm(-1) resolution was used to probe differences in wild-type and Y96F mutant P450cam (CYP101), both with and without bound camphor or styrene substrates. In the substrate-free state, the spin state equilibrium is shifted from 6-coordinate low spin (6CLS) toward more 5-coordinate high spin (5CHS) when tyrosine-96 in the substrate pocket is replaced by phenylalanine. About 25% of substrate-free Y96F mutant is 5CHS as opposed to 8% for substrate-free wild-type P450cam. Spin equilibrium constants calculated from Raman intensities indicate that the driving force for electron transfer from putidaredoxin, the natural redox partner of P450cam, is significantly smaller on styrene binding than for camphor binding. Spectral differences suggest that there is a tilt in camphor toward the pyrrole III ring on Y96F mutation. This finding is consistent with the altered product distribution found for camphor hydroxylation by the Y96F mutant relative to the single enantiomer produced by the wild-type enzyme.