Nitroreductase

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Introduction

The enzyme superfamily includes homologous enzymes, they share certain selective active site traits, and certain mechanical characteristics, but exhibit various functions. Understanding the functional differences within the superfamily is a profound problem in basic biological sciences. Do enzyme functions evolve in a sequential manner, driven by the adaptive requirements of the metabolic pathways in which they function? It is very difficult to elucidate the mechanism of functional differentiation of the enzyme superfamily, because this process occurred in an evolutionary history spanning about billions of years.

An enzyme superfamily is usually composed of many different functional families. The sequences of the functional families are very different. The pairwise sequence identity may be less than 10%, and the existing sequence information is widely dispersed. Moreover, the vast majority of enzymes in a superfamily remain uncharacterized. Superfamily usually contains more than 20,000 sequences, and the study of this large data set with significant diversity requires more advanced technology.

Later, through bioinformatics analysis, scientists solved these problems of the diversified flavin mononucleotide (FMN) dependent nitroreductase (NTR) superfamily. The NTR superfamily is an ancient superfamily with an evolutionary age of approximately 2.5 billion years, and it contains more than 20,000 sequences. This family is named after the nitro reduction reaction that was first characterized decades ago. However, in addition to nitro reduction, the NTR superfamily can also catalyze a variety of reactions, including dehydrogenation, dehalogenation, and flavin fragmentation, etc.

NTR forms an α+β fold, and non-covalently binds to flavin majority. NTRs are typically homodimers composed of two monomer subunits. Two FMN-binding active sites are formed at the dimer interface. Both monomers are important for the active sites (Figure 1A). Compared with other flavin binding proteins, dimerization is essential for FMN binding and enzymatic function in the NTR superfamily. NTR usually adopts the ping-pong bi-bi redox reaction mechanism, using nicotinamide cofactor to provide electrons to the bound FMN, and then transfer to the downstream electron acceptor in the reduction half reaction (Figure 1B and C).

Figure 1. An overview of NTR superfamily structure and reaction diversity (Akiva, E.; et al. 2017)

A Global View of Sequence Diversity Within the NTR Superfamily

In order to study the sequence diversity within the NTR superfamily, scientists collected a set of non-redundant all available sequences and structures associated with the superfamily from public databases. The data set contains 24,270 non-redundant NTR sequences that range between 150 and 1,580 aa in length. The similarity between all these sequences is calculated by using "all-vs.-all" BLAST, and SSN is used to visualize the result information.  

The SSN in Figure 2 shows nodes (circles) that present sequence sets that share >60% pairwise sequence identity; this level of similarity ensures that the sequences within a single representative node can be aligned with statistical significance and make the entire superfamily visualization. However, an identity level of 60% can also condense enzymes with different functions within a single representative node.

Figure 2. A representative SSN of the NTR superfamily (Akiva, E.; et al. 2017)

The functional diversity of the NTR superfamily remains unknown

Many studies have focused on the in-depth biochemical and structural characterization of selected few members of the superfamily, but comprehensive analysis of bioinformatics has shown that as many as 99% of the enzymes in the NTR superfamily have not been experimentally characterized. In addition, the proportion of sequences with functional and/or structural information in different subgroups is uneven. For example, few sequences in the SagB subgroup are characterized. This subgroup is large and diverse (with an average sequence identity of less than 32%), and may contain smaller "sub-subgroups (SSGs) with different substrate and catalytic specificities". Similarly, average sequence identity of less than 40% is observed in the NfsA and NfsB subgroups. At present, 18 of the 2,632 NfsB subgroup sequences and 20 of the 2,299 NfsA subgroup sequences have been characterized, approximately 1% of the total.