An alkane monooxygenase (AlkB) family in which all electron transfer partners are covalently bound to the oxygen-activating hydroxylase

Abstract

Alkane monooxygenase (AlkB) is a non-heme diiron enzyme that catalyzes the hydroxylation of alkanes. It is commonly found in alkanotrophic organisms that can live on alkanes as their sole source of carbon and energy. Activation of AlkB occurs via two-electron reduction of its diferric active site, which facilitates the binding, activation, and cleavage of molecular oxygen for insertion into an inert C-H bond. Electrons are typically supplied by NADH via a rubredoxin reductase (AlkT) to a rubredoxin (AlkG) to AlkB, although alternative electron transfer partners have been observed. Here we report a family of AlkBs in which both electron transfer partners (a ferredoxin and a ferredoxin reductase) appear as an N-terminal gene fusion to the hydroxylase (ferr_ferrR_AlkB). This enzyme catalyzes the hydroxylation of medium chain alkanes (C6-C14), with a preference for C10-C12. It requires only NADH for activity. It is present in a number of bacteria that are known to be human pathogens. A survey of the genome neighborhoods in which is it found suggest it may be involved in alkane metabolism, perhaps facilitating growth of pathogens in non-host environments.

Graphical abstract

The activity and distribution in biology of alkane monooxygenases that have a fused ferredoxin reductase and ferredoxin are reported.

Catalysts that can activate molecular oxygen and selectively hydroxylate inert C-H bonds remain an area of intense interest in part due to the chemical challenges associated with this reaction. Metalloenzymes that oxidize alkanes, including soluble and particulate methane monooxygenase (sMMO and pMMO, respectively), cytochrome P450 (CYP) and alkane monooxygenase (AlkB) carry out this reaction under ambient conditions and often show high regioselectivity. Utilizing them directly for synthetic reactions is stymied not only by their instability but also by their requirement for multiple additional proteins that serve as essential electron-transfer partners.

AlkB is the most enigmatic of the alkane monooxygenase metalloenzymes. It is a member of the class III diiron proteins, along with the mammalian fatty acid desaturases and fatty acid hydroxylases. An integral membrane protein, its three-dimensional structure has eluded determination. Mossbauer spectroscopy¹, along with mutational studies², together lead to a picture of a nitrogen-rich coordination environment in which the two iron ions are ligated to perhaps as many as nine histidines. Activation of the diiron site requires the transfer of two electrons from NADH, which are typically transferred via a reductase (AlkT) and a rubredoxin (AlkG). We recently reported the activity of an AlkB from Dietzia cinnamea with a C-terminally fused rubredoxin and described the biological contexts in which this rubredoxin-fused AlkB is found.³ Here we report an even less canonical AlkB in which the hydroxylase is N-terminally fused to both a ferredoxin reductase and a ferredoxin. Furthermore, we show that this new AlkB architecture is widespread in biology, occurring frequently in Leptospira, a genus of well-known human pathogens that cause leptospirosis. It is also found in Pseudomonas aeruginosa, an organism linked to poor-disease progression in patients with cystic fibrosis, and Stenotrophomonas maltophilia, an important agent of nosocomial infections⁴.

While the canonical octane-oxidizing system first identified in Minor Coon in 1966⁵ requires two additional proteins to serve as electron transfer partners, there is precedence for gene fusion of electron transfer proteins. Notably the mammalian fatty acid desaturases⁶ and fatty acid hydroxylases⁷ are found both with and without their proximal electron transfer partner fused.

Our interest in the three domain protein was stimulated by a report of its existence, based only on a search of microbial genomic data deposited in GenBank and the Integrated Microbial Genomes (IMG) system, in Letpospira biflexa, Limnobacteri sp. MED105, and Polaromonas napththalenivorans CJ2.⁸ We found no reports of its activity in the literature. A search of the NCBI database showed a number of proteins with similar structure, all annotated as fatty acid desaturases or as ferredoxin reductases, despite the clear appearance of an AlkB-like domain and high homology in this region to other known AlkBs and limited homology to other fatty acid desaturases (described in more detail in the SI). A cladogram of the ferr_ferrR_AlkB proteins we have identified is provided in the SI Figure S.1.

A gene encoding ferr_ferrR_AlkB from Polaromonas naphthalenivorans was codon-optimized for expression in E. coli and synthesized (IDT). This synthetic gene was inserted into a modified version of the pET15b plasmid using FastCloning.⁹ The insertion was made so that the ferr_ferrR_AlkB protein had no additional affinity purification tags (see full sequence in the SI).⁹ The protein was expressed in BL21 E. coli cells at 37 °C for four hours post induction with 200 μM IPTG. Cells were harvested, resuspended in 20 mM Tris-HCl, 150 mM NaCl, pH 7.8, 0.4 mM phenylmethylsulfonyl fluoride (PMSF) (1 ml buffer per 1 g cells) and broken open via French press, and the membrane fraction was isolated by an overnight spin at 225,000 x g. The membrane fraction was then solubilized using 20 mM Tris buffer, pH 8, with 0.18% n-Dodecyl-β-D-maltoside (DDM) (8 mL of buffer per g of membrane). Additional purification is possible using an anion exchange column (See SI Figure S.2 for more information).

Activity assays were done by adding NADH (12.7 mM) to the solubilized membrane (0.5 mL). Ferrous ammonium sulfate (1 mM) and dithionite (1 mM) were also added to ensure that the protein was fully metallated.³ Notably no additional electron-transfer proteins were required to detect enzymatic activity.

The protein was found to be active over a relatively wide range of alkanes, oxidizing hexane through tetradecane (Figure 2). A marked preference, however, was found for hydroxylating decane, undecane, and dodecane. In earlier work, we and others have associated the presence of bulky amino acids in position 55 in P putida and possibly position 56 with the preferential oxidation of alkanes of eight carbons or less.^{3, 10} In P putida these positions are occupied by W and Y. In almost all of the ferr_ferrR_AlkB sequences that we have examined, these positions are occupied by the relatively smaller amino acids V or L.

Figure 2.

Catalytic activity of ferr_ferrR_AlkB with alkanes of different carbon chain lengths, normalized to the amount of do-decanol produced when dodecane is the substrate.

Like all other AlkBs that have been previously examined, ferr_ferrR_AlkB generates a long-lived substrate radical (~10 nanoseconds) when oxidizing the radical clock substrate norcarane (Figure 3, additional information on how the radical lifetime was calculated is provided in the SI).^11–17 All AlkBs that have been previously examined catalyze the epoxidation of terminal alkenes with similar if not better efficiency than they hydroxylate alkanes.^18,19 Ferr_ferrR_AlkB in contrast does not catalyze the epoxidation of alkenes (Figure S.3). Epoxidation may be tied to structural features in the active site, such as those that provide sufficient mobility for the high valent species to interact with both the terminal and β positions.³ Ferr_ferrR_AlkB’s inability to epoxidize alkenes likely points to an intriguing structural difference between the more canonical AlkBs and members of this new family.³

Figure 3.

Overlay of live and control reaction showing the oxidation of norcarane catalyzed by ferr_ferrR_AlkB. The two mechanistically significant products are identified, along with the product formed from oxidation at the mechanistically insignificant 3-position. The peak at 7.85 is not norcarane-derived.

AlkBs are widely recognized as catalyzing the initial step in the mineralization of alkanes. First identified in Pseudomonas oleovorans (now known as P. putida GPo1), the alkB gene in P. putida GPo1 is part of an OCT operon that contains numerous other genes required to oxidize alkanes and prepare them to enter the fatty acid cycle.^20,21 Searching the gene region (+/− 10,000 bp) around the ferr_ferrR_alkB, numerous cases were found where the ferr_ferrR_alkB gene appears at the start of a series of genes that code for one or more of the following: an alcohol dehydrogenase; a fatty acetyl-CoA synthase; an aldehyde dehydrogenase. Fatty acetyl-CoA synthases are a key part of the β-oxidation pathway and have been shown to be upregulated when alkane metabolizing organisms are grown on alkanes²², suggesting that this set of genes constitutes a non-canonical alkane oxidation pathway. No instances of electron transfer proteins near the ferr_ferrR_alkB gene were found, again consistent with the evidence provided here that this protein contain all of the electron-transfer partners required for activity. A detailed picture of the gene neighborhood analysis is provided in the SI (Figure S.4).

In conclusion, we have established the functional relevance of the three-domain AlkB. This is the first AlkB identified that uses ferredoxin reductase and ferredoxin as electron transfer partners and the first AlkB in which these electron transfer partners are fused to the monooxygenase. We have demonstrated the prevalence of the three-domain AlkB in biology and note that it is consistently annotated as either a fatty acid desaturase (despite the alkane hydroxylase domain clearly having the features that distinguish AlkB from a fatty acid desaturase, ferredoxin reductase, or flavin reductase). The ability to activate three domain AlkB with only NADH will facilitate its use in synthetic chemistry. Work is on-going to determine the structure of this member of the class III diiron hydroxylase family. Microbiology studies, outside the scope of our expertise, will be required to elucidate its functional role in biology.

Figure 1.

Typical arrangement of AlkB-associated alkane oxidizing proteins. AlkL is an outer membrane transporter that allows alkanes to enter the cell. AlkB is an integral membrane protein that spans the cytoplasmic membrane and catalyzes the hydroxylation of alkanes using molecular oxygen. AlkG and AlkT are the rubredoxin and reductase, respectively, that shuttle electrons to AlkB. AlkJ catalyzes the oxidations of alcohols to aldehydes and AlkH catalyzes the oxidation of aldehydes to carboxylic acids, after which they are acetylated with acetyl CoA and then entered into the fatty acid cycle. Inset shows two other arrangements of electron transfer partners that have been found with AlkB. A rubredoxin is C-terminally fused to AlkB in the two-domain structure. In the three-domain structure, reported here, ferredoxin reductase and ferredoxin are both N-terminally fused to AlkB. The blue arrows indicate the flow of electrons and the red arrows indicate the flow of substrate to products

Highlights.

• Alkane monooxygenases with fused ferredoxin reductase and ferredoxin exist in nature.

• These three-domain AlkBs catalyze the hydroxylation of alkanes and require only NADH for activity.

• The three-domain AlkBs are misannotated in protein databases, despite being easily identified as an alkane hydroxylase.