Microorganisms play indispensable roles in the degradation and detoxification of polycyclic aromatic hydrocarbons (PAHs) in the environment 12. The initiation of the aerobic microbial degradation of PAHs is an oxidative attack 34. The enzymes that catalyze insertion of molecular oxygen into aromatic benzene rings are termed oxygenases 5. They require transition metals, such as iron and heme, as catalytic centers. Oxygenases that utilize non-heme Fe(II) are called Rieske-type non-heme iron aromatic ring-hydroxylating oxygenase (RHO) whereas others that use heme are cytochrome P450s 67. The term RHO is used herein to denote the Rieske-type non-heme iron ring-hydroxylating oxygenase.

Although RHOs mostly use NAD(P)H as an electron donor and catalyze the same oxygenation reaction, they are remarkably diverse with respect to their structure 348. RHOs are multi-component enzymes of two or three protein components consisting of an electron transport chain (ETC) and an oxygenase. Oxygenase components are either homo- (α_n) or hetero-oligomers (α_nβ_n) and in each case, the α subunit, called large subunit, contains two conserved regions, a Rieske [2Fe-2S] center and non-heme mononuclear iron. The α subunits are known to be the catalytic components involved in the transfer of electrons to oxygen molecules. The ETC that transfers reducing equivalents from NAD(P)H to the oxygenase components consists of either a flavoprotein reductase or a flavoprotein reductase and a ferredoxin 34. An interaction between oxygenase and ETC components is required for the enzyme system to transfer electrons from the electron donor to aromatic hydrocarbon electron acceptor. The RHO enzyme system has been extensively studied in many different microorganisms since the initial reaction mostly determines the aromatic substrate for degradation 9101112131415.

Classification of RHOs is essentially an effort to organize the information into a system that is useful for understanding the relationship between various aspects of sequence, structure, function and evolution. A three-class system (class I, II and III) was initially instituted by Batie et al. 16. Based on the number of constituent components and the nature of the redox centers, this classification was able to give systematic information about RHOs. We will refer to this approach as "the traditional classification". With the recent tremendous accumulation of new sequence information on RHOs, there is a current need for a new classification strategy that can transform the multitude of complex data into useful organized information. In this regard, computational phylogenetic analysis of molecular sequence was imperative, which we term "the phylogenetic classification". Several challenges have been introduced using this method. Werlen et al. 17 grouped RHOs into four families based on substrate specificities and sequence alignments with associated distance calculations. This classification emphasized the structure-function relationship of the oxygenase component. However, some RHOs appear not to fit in this scheme probably because of the small RHO sample pool which resulted in the partial observation. Nam et al. 18 also proposed a clustering system based on the homology of the amino acid sequences of terminal oxygenase components. This classification system was more inclusive and well reflected the phylogenetic affiliation among RHOs.

In recent years, we characterized 3 oxygenases, NidAB, NidA3B3 and PhtAaAb, from M. vanbaalenii PYR-1 involved in the oxidation of aromatic hydrocarbons 9101519. Phylogenetic analysis of these oxygenase components, NidA, NidA3 and PhtAa, showed that they clustered with a new group of α subunits found in Nocardioides, Rhodococcus, Terrabacter, Arthrobacter and other Mycobacterium spp. 202122232425. Interestingly, RHO genes found in this group of bacteria appear to have features in common; genes for ETC components are not always closely positioned with oxygenase genes (genetic discreteness) and limited numbers of ferredoxin and reductase components are shared by multiple oxygenases (numerical imbalance). Interestingly, ferredoxin components, that are compatible with the oxygenase enzymes from this bacterial group, were often identified to be a [3Fe-4S] cluster type 202223. The [3Fe-4S] type of ferredoxin has been recently introduced as an ETC component for RHO enzyme systems, the other ferredoxin components being the [2Fe-2S] type. In fact, NidAB and NidA3B3 from PYR-1 were also shown to be compatible with the [3Fe-4S]-type ferredoxin, PhtAc and ferredoxin reductase (PhtAd) 19. However, the classification system proposed by Batie et al. and Nam et al. can not explain these new RHO features, the [3Fe-4S]-type ferredoxin, genetic discreteness of ETC components and the numerical imbalance between oxygenase and ETC component. In addition, considering that RHOs are multi-component enzyme systems, ETC components along with oxygenases components are also necessary for their functional understanding. Therefore, the new classification system was launched with the aim of analyzing the RHO components as a whole. The classification system not only systematically incorporates each component data but also basically reflects the previous "traditional" and "phylogenetic" classification.

Construction of the new classification scheme was accomplished as shown in the flowchart (Figure 1). Briefly, we first analyzed a total of 130 RHO enzymes in which 25 well-characterized RHO enzymes were selected as standards to test our hypothesis for the proposed classification system (Table 1). From the sequence analysis of ETC components of the standard RHO samples, we extracted the classification keys that reflect the phylogenetic relationship among ETC components, which turned into the grouping criteria in the following classifications. Next, oxygenases were phylogenetically classified with the classification keys obtained from ETC analyses. Finally we constructed the new systematic classification through statistical justification and tested its applicability with 38 RHO samples.

In many cases, RHO oxygenases have not been identified together with ETC components, thus it has been a challenging task to find a cognate ETC partner that can function in cooperation with these "incomplete" oxygenase components 93536373839. Since some ETCs are interchangeable with each other and can be often replaced by outside ETC sources and it is the terminal oxygenase component that has the catalytic active site, less emphasis was given to ETC components in Nam's classification. For example, DitA from P. abietaniphila BKME-9 and NidA from Rhodococcus sp. strain I24 were clustered in group III together with NahAc from Pseudomonas sp. NCIB9816-4 and PahAc from P. putida OUS82. However, these two oxygenases, DitA and NidA, which lacked ETC information (only ferredoxin for DitA is known), initially formed a group different from that of NahAc and PahAc in the phylogenetic classification route, being classified as type V that represents the three-component systems consisting of an oxygenase, a [3Fe-4S]-type ferredoxin and a GR-type reductase. Whereas the NahAc and PahAc were classified as type III which consists of an oxygenase, a [2Fe-2S]-type ferredoxin and an FNR_N-type reductase. The discrepancy between these two classification's results comes from ETC information that reflects the relationship among oxygenases. The classification in Nam's scheme was based on pairwise sequence homology of oxygenase components, in which four percentage sets, 0–14, 15–24, 25–34 and 35–99%, were used. In the new classification, however, the grouping criterion for oxygenase components, which is a PD value of 0.62, was objectively determined based on ETC information. This PD value finally determines the two oxygenases, DitA and NidA, differently from the NahAc and PahAc. The results clearly show that information on oxygenases whose ETC components are not available can be used to draw ETC data in our classification system.

This new classification system provides insights into the evolutionary changes and relationships between oxygenase and ETC components. Seven oxygenase components, BphA1 [a-f] and XylX from N. aromaticivorans F199 37 are of particular interest in this context. Although other ETC components are maybe involved, those oxygenases from F199, which were found to be scattered throughout several gene clusters, seem to function with a limited number of ETC components. Two ferredoxins (Rieske- [2Fe-2S]-type BphA3 and plant- [2Fe-2S]-type XylT) and two reductases (GR-type BphA4 and FNR-type XylM) have been identified on the aromatic catabolic plasmid (pNL1) of F199. These oxygenases were classified as type II (XylX), IIIA (BphA1 [a,b,f]) and IV (BphA1 [c-e]) (Figure 8). Although there is no functional data available directly from the strain F199, genetic information with functional evidence have been reported from other sphingomonads which matches with those of F199. For example, sets of genes for an oxygenase from Sphingobium sp. P2 (AhdA1 [a-e]) 36, S. yanoikuyae B1 (BphA1c and XylX) 4041 and Sphingobium sp. CHY-1 (PhnA1a and PhnA1b) 42 were reported to have the same gene arrangement with 63–97% sequence identity when compared to F199. Consequently, they were classified as the same types with those from F199; type II for XylX (B1), type IIIαβ for AhdA1 [a,b] (P2), PhnA1a (CHY-1) and type IV for BphA1c (B1), AhdA1 [c-e] (P2) and PhnA1b (CHY-1). We found that, without an exception, they were experimentally shown to use type IV ETC (all named as BphA3A4) that consists of [2Fe-2S]-type ferredoxin and GR-type reductase (Figure 6). These ETCs are highly homologous (> 79%) to the BphA3A4 from F199. Therefore, it is reasonable to predict that the 7 oxygenase components (BphA1 [a-f] and XylX) from F199 might be sharing the same type of ETC, BphA3A4. This implies that the limited ETC (BphA3A4) probably makes for strong selective pressure, favoring oxygenase components that can rapidly adapt by increasing their tolerance toward alternative ETC. This adaptation should be accompanied by the change of genetic information, which suggesting that RHO enzymes are probably evolving under the direction of selective pressure derived from ETCs. In fact, although BphA1c from F199 was clustered in type IV, it intersected with type III, the group of 2-hydroxybenzoate 5-salicylate hydroxylase (HybABCD) from P. aeruginosa JB2 (Figure 8). It suggests that BphA1c from F199 is probably evolving under the selective pressure derived from limited ETC. The XylX from the strain B1 is also worth noting, XylX belongs to type II, the two-component RHO enzymes, which normally functions with an FNR-type reductase. However, it was unexpectedly shown to be compatible with BphA3A4 4041. Although both ferredoxins from type III and IV are [2Fe-2S]-type, the ferredoxin component (BphA3) from B1 seems phylogenetically inclined to be branched with ferredoxins which can complement oxygenases belong to type IIIαβ. The inclination of the ferredoxin component (BphA3) toward type IIIαβ also can be seen in the RHO enzyme from Cycloclasticus sp. A5, of which PhnA1 and PhnA3 components showed high identities over 62% with those (BphA1f and BphA3, respectively) from the strain F199 43. In this case, the oxygenase (PhnA1) belongs to type IIIαβ with the ETC components consist of a [2Fe-2S]-type ferredoxin (PhnA3) and an FNR_N-type reductase (PhnA4). Taken together, the 7 oxygenases from F199, belonging to type II, IIIA and IV, were classified by our system to work with the three different ETC groups, FNR_N-type reductase, [2Fe-2S]-type ferredoxins/FNR_N-type reductase and [2Fe-2S]-type ferredoxins/GR-type reductase, respectively. However, all those oxygenases are assumed to function with [2Fe-2S]-type ferredoxin and GR-type reductase. From these observations question may arise about the degree of specificity in recognition between redox partners.

RHO pool is believed to respond dynamically to the environmental transitions such as substrate changes. In addition, relative tolerance between oxygenase and ETC components is also thought to be one of the important selective forces in evolution. In this context, even though little is known as to the role of ferredoxin component under selective pressure, it seems likely to be deeply involved in evolutionary changes. A possible benefit for using ferredoxin comes from its potential to promptly enhance the tolerance toward new redox partners. Cognizant of "the shorter, the faster and more effective", because of the relatively short sequence and simple structure of ferredoxin than that of reductase component, it has an evolutionary merit to adapt rapidly to dynamic environments. Hence, it might be an attractive hypothesis that ferredoxin has been evolutionarily chosen as a buffer between reductase and oxygenase component for rapid adaptation toward selective force. It might also be a strong driving force to affect the direction of evolution from two-component RHO to/toward three-component. Considering this two-way communication flow in RHO system, the adoption of ferredoxin as a new redox partner must have led to the changes in other partners, reductase and oxygenase, in RHO system. In this context, type III RHO seems likely to be a living fossil that has recorded evolutionary changes because they show transitional properties from two-component to three-component system. Two-component systems for type I and II share FNR-type reductase, FNR_C- and FNR_N-type, respectively, which consist of three domains (flavin, NAD and [2Fe-2S] binding). On the other hand, three-component systems for type IV and V, except for type III, share a GR-type reductase, which have no [2Fe-2S] cluster (Figure 2 and Figure 6). A [2Fe-2S] cluster of reductases separates the two-component system (type I and II) from the three-component system (type IV and V). It strongly demonstrates that the joining of ferredoxin to the two-component RHO system was probably the start of the type III three-component structure. During evolution, the type III FNR_N-type reductases might have been gradually changed toward type IV and V GR-type reductases, although it is not sure whether it was recruited from other systems. In some cases, it might also be possible under extreme conditions that type I and II RHO systems evolved directly to type IV (or V) as seen in the XylX from S. yanoikuyae B1. Taken together, it may be postulated that RHOs are evolving under selective pressures from type I and II (two-component system) toward type IV and V (three-component), in which this change moves toward efficiency and keeps going continuously under dynamic environments.

In this study, we have developed a new classification system to classify bacterial Rieske non-heme iron aromatic ring-hydroxylating oxygenases. The new classification system presented in this study not only reflects sequence information but also interactions between RHO enzyme components. The system is characterized by the features that include the following. First, the new classification system analyzes RHO enzymes as a whole, in which information on each RHO components is organized into a system that is useful for the understanding of various aspects with respect to sequence, structure and biochemical function. Second, our new classification system is not static but responds dynamically to the growing pool of RHO enzymes. As standard RHO samples increase, the classification system evolves, which gets more stable and objective and could increase its coverage of many other RHO enzymes. Third, our classification can be applied reliably to the classification of incomplete RHOs. Fourth, the classification has direct applicability to experimental work. Our classification can provide the information about cognate ETC partners for oxygenase catalytic activity. Fifth, the system provides new insights into the evolution of RHO systems based on enzyme interaction.

PAH, polycyclic aromatic hydrocarbons; RHO, Rieske-type non-heme iron aromatic ring-hydroxylating oxygenase; ETC, electron transport chain; PD, pairwise distance; NJ, neighbor-joing; CD, conserved domain; NR, nonredundant; GR, glutathione reductase; FNR, ferredoxin-NADP⁺ reductase.

All authors participated in the design of the study and writing of the manuscript. CEC directed the whole research and critically revised the manuscript. All authors read and approved the final manuscript.