Our previous rapid-scanning stopped-flow studies of the reaction of substrate-free cytochrome P450cam with peracids [T. Spolitak, J.H. Dawson, D.P. Ballou, J. Biol. Chem. 280 (2005) 20300-20309; J. Inorg. Biochem. 100 (2006) 2034-2044; J. Biol. Inorg. Chem. 13 (2008) 599-611] spectrally characterized compound I (ferryl iron plus a porphyrin pi-cation radical (Fe(IV)O/Por(.+))), Cpd ES, and Cpd II (Fe(IV)O/Tyr() or Fe(IV)O). We now report that reactions of CYP153A6 with peracids yield all these intermediates, with kinetic profiles allowing better resolution of all forms at pH 8.0 compared to similar reactions with WT P450cam. Properties of the reactions of these higher oxidation state intermediates were determined in double-mixing experiments in which intermediates are pre-formed and ascorbate is then added. Reactions of heptane-bound CYP153A6 (pH 7.4) with mCPBA resulted in conversion of P450 to the low-spin ferric form, presumably as heptanol was formed, suggesting that CYP 153A6 is a potential biocatalyst that can use peracids with no added NAD(P)H or reducing systems for bioremediation and other industrial applications.

This review focuses on the role and distribution in the environment of alkane hydroxylases and their (potential) applications in bioremediation and biocatalysis. Alkane hydroxylases play an important role in the microbial degradation of oil, chlorinated hydrocarbons, fuel additives, and many other compounds. Environmental studies demonstrate the abundance of alkane degraders and have lead to the identification of many new species, including some that are (near)-obligate alkanotrophs. The availability of a growing collection of alkane hydroxylase gene sequences now allows estimations of the relative abundance of the different enzyme systems and the distribution of the host organisms.

The hydroxylation of alkane molecules, especially at terminal positions, is a challenging reaction. Enzymes that catalyze this reaction could be used to produce high-value compounds from aliphatic and alkyl-substituted substrates. However, until a few years ago, all known alkane hydroxylating enzymes were membrane-bound, and difficult to use. Recently, three bacterial P450 enzymes of the ( soluble) CYP101 and CYP102 families were engineered to hydroxylate alkanes, but even after extensive efforts hydroxylation was mainly at sub-terminal positions. More recently, a new soluble P450 family (CYP153) was identified and characterized, which activates the terminal position of alkanes and alkyl-substituted compounds with very high regio-selectivity. The use of CYP153s in biotechnological applications is now being explored.

Several cytochrome P450 alkane hydroxylases of the CYP153 family were recently identified and functionally expressed in Pseudomonas putida GPo12 [van Beilen JB, Funhoff EG, van Loon A, Just A, Kaysser L, Bouza M, et al. Cytochrome P450 alkane hydroxylases of the CYP153 family are common in alkane-degrading eubacteria lacking integral membrane alkane hydroxylases. Appl Environ Microbiol 2006;72:59–65]. Eight recombinants containing divergent CYP153's were cultivated on a 1.5–1.8L scale with octane or decane/dodecane as sole carbon source. Growth rates between 0.06 and 0.23h−1 were obtained and analysis of cell free extracts showed that the CYP153's were expressed to a maximum of 11% of total protein. The hydroxylation activities towards various substrates translate to maximal CYP153 turnover numbers between <0.1 and 58min−1. Aliphatic alkanes were exclusively hydroxylated on terminal carbon atoms. Epoxidation experiments with styrene, octene and cyclohexene showed turnover numbers that were slightly lower than the hydroxylation activities, with the exception of CYP153A11 and CYP153A14. The results provide insight in structural features that determine the substrate specificity of these enzymes.

ABSTRACT The first and key step in alkane metabolism is the terminal hydroxylation of alkanes to 1-alkanols, a reaction catalyzed by a family of integral-membrane diiron enzymes related to Pseudomonas putida GPo1 AlkB, by a diverse group of methane, propane, and butane monooxygenases and by some membrane-bound cytochrome P450s. Recently, a family of cytoplasmic P450 enzymes was identified in prokaryotes that allow their host to grow on aliphatic alkanes. One member of this family, CYP153A6 from Mycobacterium sp. HXN-1500, hydroxylates medium-chain-length alkanes (C 6 to C 11 ) to 1-alkanols with a maximal turnover number of 70 min −1 and has a regiospecificity of ≥95% for the terminal carbon atom position. Spectroscopic binding studies showed that C 6 -to-C 11 aliphatic alkanes bind in the active site with K d values varying from ∼20 nM to 3.7 μM. Longer alkanes bind more strongly than shorter alkanes, while the introduction of sterically hindering groups reduces the affinity. This suggests that the substrate-binding pocket is shaped such that linear alkanes are preferred. Electron paramagnetic resonance spectroscopy in the presence of the substrate showed the formation of an enzyme-substrate complex, which confirmed the binding of substrates observed in optical titrations. To rationalize the experimental observations on a molecular scale, homology modeling of CYP153A6 and docking of substrates were used to provide the first insight into structural features required for terminal alkane hydroxylation.

Several strains that grow on medium-chain-length alkanes and catalyze interesting hydroxylation and epoxidation reactions do not possess integral membrane nonheme iron alkane hydroxylases. Using PCR, we show that most of these strains possess enzymes related to CYP153A1 and CYP153A6, cytochrome P450 enzymes that were characterized as alkane hydroxylases. A vector for the polycistronic coexpression of individual CYP153 genes with a ferredoxin gene and a ferredoxin reductase gene was constructed. Seven of the 11 CYP153 genes tested allowed Pseudomonas putida GPo12 recombinants to grow well on alkanes, providing evidence that the newly cloned P450s are indeed alkane hydroxylases.

Proteolysis of single polypeptide mammalian purple acid phosphatases (PAPs) results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and/or His92. While Asn91 is a ligand to the divalent metal of the mixed‐valent di‐iron center, the role of His92 in the catalytic mechanism is unknown. Site‐directed mutagenesis of His92 was performed to examine the role of this residue in single polypeptide PAP. Conversion of His92 into Ala, which eliminates polar interactions of this residue with the active site, resulted in a 10‐fold decrease in catalytic activity at the optimal pH. Conversely, conversion of this residue into Asn, which cannot function as either a proton donor or acceptor, but can provide hydrogen–bonding interactions, resulted in a three‐fold increase in activity at the optimal pH. Both mutant enzymes had more acidic pH optima, with pKes,1 values consistent with the involvement of an iron(III) hydroxide unit or a hydroxide in the second coordination sphere in catalysis. These results, together with EPR data, support a role of His92 in positioning either the nucleophile or the substrate, rather than directly in acid or base catalysis. The existence of an extensive hydrogen‐bonding network that could fine‐tune the position of His92 is consistent with this proposal.

As highly reduced hydrocarbons are abundant in the environment, enzymes that catalyze the terminal or subterminal oxygenation of alkanes are relatively easy to find. A number of these enzymes have been biochemically characterized in detail, because the potential of alkane hydroxylases to catalyze high added-value reactions is widely recognized. Nevertheless, the industrial application of these enzymes is restricted owing to the complex biochemistry, challenging process requirements, and the limited number of cloned and expressed enzymes. Rational and evolutionary engineering approaches have started to yield more robust and versatile enzyme systems, broadening the alkane oxygenase portfolio. In addition, metagenomic approaches provide access to many novel alkane oxygenase sequences.

To date, most spectroscopic studies on mammalian purple acid phosphatases (PAPs) have been performed at a single pH, typically pH 5. The catalytic activity of these enzymes is, however, pH dependent, with optimal pH values of 5.5–6.2 (depending on the form). For example, the pH optimum of PAPs isolated as single polypeptides is around pH 5.5, which is substantially lower that of proteolytically cleaved PAPs (ca. pH 6.2). In addition, the catalytic activity of single polypeptide PAPs at their optimal pH values is four to fivefold lower than that of the proteolytically cleaved enzymes. In order to elucidate the chemical basis for the pH dependence of these enzymes, the spectroscopic properties of both the single polypeptide and proteolytically cleaved forms of recombinant human PAP (recHPAP) and their complexes with inhibitory anions have been examined over the pH range 4 to 8. The EPR spectra of both forms of recHPAP are pH dependent and show the presence of three species: an inactive low pH form (pHpK a,2). The pK a,1 values observed by EPR for the single polypeptide and proteolytically cleaved forms are similar to those previously observed in kinetics studies. The spectroscopic properties of the enzyme–phosphate complex (which should mimic the enzyme–substrate complex), the enzyme–fluoride complex, and the enzyme–fluoride–phosphate complex (which should mimic the ternary enzyme–substrate–hydroxide complex) were also examined. EPR spectra show that phosphate binds to the diiron center of the proteolytically cleaved form of the enzyme, but not to that of the single polypeptide form. EPR spectra also show that fluoride binds only to the low pH form of the enzymes, in which it presumably replaces a coordinated water molecule. The binding of fluoride and phosphate to form a ternary complex appears to be cooperative.

The kinetics and spectroscopic properties of the single polypeptide and proteolytically cleaved form of recombinant Fe3+Fe2+ human purple acid phosphatase (recHPAP) exhibit significant differences, primarily due to a difference in pKes,1 (the value of an acid dissociation constant of the ES complex). These differences are due to the presence or absence, respectively, of an interaction between an aspartate residue in an exposed loop of the protein and one or more active site residues. To further explore the origin of these differences, the ferrous ion of recHPAP has been replaced by zinc. Analysis of the reconstituted Fe3+Zn2+recHPAP reveals an unexpected catalytic activity versus pH profile, in that the optimal pH is 6.3, similar to that of the proteolytically cleaved form (6.5). Moreover, replacement of the ferrous ion by zinc increases the turnover number more than 10-fold; the pKes values are also shifted as expected for the change in the divalent metal ion. Although the EPR spectra of both single polypeptide and proteolytically cleaved Fe3+Zn2+-recHPAP are independent of pH over the range 4.5–6.2, the visible spectrum of Fe3+Zn2+-recHPAP is pH dependent. These results suggest that the properties and environment of the divalent metal are important in determining the catalytic properties of mammalian PAPs, and in particular that a solvent molecule coordinated to the divalent metal ion may play a critical role in the catalytic cycle of these enzymes.

Proton NMR spectra of FeIII-FeII recombinant single polypeptide human PAP (recHPAP) have been measured at, above, and below its pH optimum, as have the spectra of inhibited forms containing fluoride and phosphate, analogues of the substrates hydroxide and phosphate esters, respectively. The results demonstrate that binding of inhibitory anions to the dinuclear mixed-valent site of recHPAP is controlled by protonation of a ligand to the dinuclear center. Thus, the group that is responsible for pKa,1 in the enzymatic activity versus pH profile functions as a "gatekeeper", whose protonation state controls anion binding to the mixed-valent dinuclear site. The correlation between the pKa values observed in kinetics studies and for the spectroscopic changes strongly suggests that this group is the nucleophilic hydroxide that attacks the phosphate ester substrate.

Abstract Recombinantt human purple acid phosphatase (recHPAP) provides a convenient experimentall system for assessing the relationship between molecular structure and enzymaticc activity in mammalian purple acid phosphatases (PAPs). RecHPAP is a single polypeptidee protein with properties similar to those of uteroferrin (Uf) and other PAPs isolatedd as single polypeptide chains, but its properties differ significantly from those of bovinee spleen PAP (BSPAP) and other PAPs isolated as proteolytically "cl ipped" forms. Incubationn of recHPAP with trypsin results in proteolytic cleavage in an exposed region nearr the active site. The product is a tightly associated two-subunit protein whose collectivee spectroscopic and kinetics properties resemble those of BSPAP. These results demonstratee that the differences in spectroscopic and kinetics properties previously reportedd for mammalian PAPs are the result of proteolytic cleavage. Masss spectrometry shows that a 3-residue segment, D-V-K, within the loop region is excisedd by trypsin. This finding suggests that interactions between residues in the excisedd loop and one or more of the groups that participate in catalysis are lost or alteredd upon proteolytic cleavage. Analysis of available structural data indicates that the mostt important such interaction is that between Asp l46 in the exposed loop and active sitee residues Asn91 and His92. Loss of this interaction should result in both an increase inn the Lewis acidity of the Fe ion and an increase in the nucleophilicity of the Feboundd hydroxide. Proteolytic cleavage thus could constitute a potential physiological mechanismm for regulating the activity of PAP in vivo.

Recombinant human purple acid phosphatase (recHPAP) provides a convenient experimental system for assessing the relationship between molecular structure and enzymatic activity in mammalian purple acid phosphatases (PAPs). recHPAP is a monomeric protein with properties similar to those of uteroferrin (Uf) and other PAPs isolated as single polypeptide chains, but its properties differ significantly from those of bovine spleen PAP (BSPAP) and other PAPs isolated as proteolytically “clipped” forms. Incubation of recHPAP with trypsin results in proteolytic cleavage in an exposed region near the active site. The product is a tightly associated two-subunit protein whose collective spectroscopic and kinetics properties resemble those of BSPAP. These results demonstrate that the differences in spectroscopic and kinetics properties previously reported for mammalian PAPs are the result of proteolytic cleavage. Mass spectrometry shows that a three-residue segment, D-V-K, within the loop region is excised by trypsin. This finding suggests that important interactions between residues in the excised loop and one or more of the groups that participate in catalysis are lost or altered upon proteolytic cleavage. Analysis of available structural data indicates that the most important such interaction is that between Asp 146 in the exposed loop and active-site residues Asn 91 and His 92. Loss of this interaction should result in both an increase in the Lewis acidity of the FeII ion and an increase in the nucleophilicity of the FeIII-bound hydroxide ion. Proteolytic cleavage thus constitutes a potential physiological mechanism for regulating the activity of PAP in vivo.

Mammalian purple acid phosphatases (PAPs) can be divided into two groups, which exhibit distinct spectroscopic and kinetics properties: PAPs that consist of a single 36 kDa polypeptide, and PAPs that have undergone limited proteolysis to give two fragments with masses of 16 and 20 kDa, respectively. Proteolysis results in an increase in enzymatic activity, an increase in the optimal pH for activity, and a change in the g(z) value of the characteristic EPR spectrum of the mixed-valence binuclear iron center. It has been proposed that these changes are due to the loss of interactions between Asp146 in an exposed loop region and active site residues upon proteolysis. In the present study, site-directed mutagenesis of Asp146 in recombinant rat bone PAP (recRPAP) has confirmed this hypothesis. Conversion of Asp146 into Ala, which eliminates the interaction of the side chain with the active site, resulted in an enzyme with properties typical of PAPs isolated in proteolytically cleaved forms. The Asp146Asn and Asp146Glu mutants were also prepared and examined to assess the effects of altered electrostatic interactions and side-chain length. Limited proteolysis of all three mutant enzymes with cathepsin L resulted in a significant increase in catalytic activity. Thus, although the interaction between Asp146 and (an) active site residue(s) is the major factor responsible for the low catalytic activity of uncleaved PAPs, other interactions are also important. Since both p-nitrophenyl phosphate and osteopontin, a potential in vivo substrate, show the same level of activation, the observed increase in catalytic activity upon proteolysis is likely to be due to electrostatic rather than steric effects. EPR spectra of FeZn-recRPAP before and after cleavage by cathepsin L suggest that cleavage primarily affects the divalent metal site. The observation that pK(es,1) is also sensitive to changes at the divalent site is consistent with the proposal that the nucleophilic hydroxide is that bridging the divalent and trivalent metals.