Genomic Enzymology: Web Tools for Leveraging Protein Family Sequence–Function Space and Genome Context to Discover Novel Functions

Abstract

The exponentially increasing number of protein and nucleic acid sequences provides opportunities to discover novel enzymes, metabolic pathways, and metabolites/natural products, thereby adding to our knowledge of biochemistry and biology. The challenge has evolved from generating sequence information to mining the databases to integrating and leveraging the available information, i.e., the availability of “genomic enzymology” web tools. Web tools that allow identification of biosynthetic gene clusters are widely used by the natural products/synthetic biology community, thereby facilitating the discovery of novel natural products and the enzymes responsible for their biosynthesis. However, many novel enzymes with interesting mechanisms participate in uncharacterized small-molecule metabolic pathways; their discovery and functional characterization also can be accomplished by leveraging information in protein and nucleic acid databases. This Perspective focuses on two genomic enzymology web tools that assist the discovery novel metabolic pathways: (1) Enzyme Function Initiative-Enzyme Similarity Tool (EFI-EST) for generating sequence similarity networks to visualize and analyze sequence–function space in protein families and (2) Enzyme Function Initiative-Genome Neighborhood Tool (EFI-GNT) for generating genome neighborhood networks to visualize and analyze the genome context in microbial and fungal genomes. Both tools have been adapted to other applications to facilitate target selection for enzyme discovery and functional characterization. As the natural products community has demonstrated, the enzymology community needs to embrace the essential role of web tools that allow the protein and genome sequence databases to be leveraged for novel insights into enzymological problems.

‘Democratized’ genomic enzymology web tools for functional assignment

“…However, the use of Pythoscape requires knowledge of programming as well access to a computer cluster. The Enzyme Function Initiative (EFI [15]) developed and provides a “democratized,” i.e., publicly accessible, web tool for generating SSNs, the EFI-Enzyme Similarity Tool (EFI-EST; https://efi.igb.illinois.edu/efi-est/ [12,16]). Using EFI-EST, SSNs can be constructed for Pfam and/or InterPro families (and/or user-defined sets of sequences).…”

“…The EFI also offers the democratized EFI-Genome Neighborhood Tool (EFI-GNT; https://efi.igb.illinois.edu/efi-gnt/[12]) that provides statistical analysis of genome context for bacterial and fungal members of protein families to identify possible functional linkage, e.g., metabolic pathways. Each sequence in an input SSN is used to query its genome neighborhood.…”

Identification of a Functionally Unique Family of Penicillin-Binding Proteins

“…To identify other possible low molecular weight transpeptidases, we generated a sequence similarity network (SSN) for the Peptidase S11 family (Figures 2, S2). 8 The alignment analysis was restricted to only the catalytic domain of the family members. We found that Sa PBP4 and PBPs from at least 40 other Gram-positive bacteria (Table S2) clustered into two related groups that were separated from a much larger group containing known hydrolases, including E. coli PBP5 ( Ec PBP5), a well-characterized carboxypeptidase.…”

Description of a widespread bacterial secretion system with chemically diverse substrates

“…We sought to gain more quantifiable information about whether sub-families of DUF560 homologs exist and whether DUF560 are enriched among mucosal symbionts. A sequence similarity network of DUF560 homologs was generated using the Enzyme Function Initiative (EFI) [33][34][35][36] (Fig. 2) and manually annotated using publicly available database entry information to highlight either the environmental source ( Fig.…”