Evolution of Enzyme Superfamilies: Comprehensive Exploration of Sequence-Function Relationships.

The sequence and functional diversity of enzyme superfamilies have expanded through billions of years of evolution from a common ancestor. Understanding how protein sequence and functional "space" have expanded, at both the evolutionary and molecular level, is central to biochemistry, molecular biology, and evolutionary biology. Integrative approaches that examine protein sequence, structure, and function have begun to provide comprehensive views of the functional diversity and evolutionary relationships within enzyme superfamilies. In this review, we outline the recent advances in our understanding of enzyme evolution and superfamily functional diversity. We describe the tools that have been used to comprehensively analyze sequence relationships and to characterize sequence and function relationships. We also highlight recent large-scale experimental approaches that systematically determine the activity profiles across enzyme superfamilies. We identify several intriguing insights from this recent body of work. First, promiscuous activities are prevalent among extant enzymes. Second, many divergent proteins retain "function connectivity" via enzyme promiscuity, which can be used to probe the evolutionary potential and history of enzyme superfamilies. Finally, we discuss open questions regarding the intricacies of enzyme divergence, as well as potential research directions that will deepen our understanding of enzyme superfamily evolution.

Toward a high-throughput screening platform for directed evolution of enzymes that activate genotoxic prodrugs.

Engineering of enzymes to more efficiently activate genotoxic prodrugs holds great potential for improving anticancer gene or antibody therapies. We report the development of a new, GFP-based, high-throughput screening platform to enable engineering of prodrug-activating enzymes by directed evolution. By fusing an inducible SOS promoter to an engineered GFP reporter gene, we were able to measure levels of DNA damage in intact Escherichia coli and separate cell populations by fluorescence activating cell sorting (FACS). In two FACS iterations, we were able to achieve a 90,000-fold enrichment of a functional prodrug-activating nitroreductase from a null library background.

Targeted mutagenesis of the Vibrio fischeri flavin reductase FRase I to improve activation of the anticancer prodrug CB1954.

Phase I/II cancer gene therapy trials of the Escherichia coli nitroreductase NfsB in partnership with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide] have indicated that CB1954 toxicity is dose-limiting at concentrations far below the enzyme K(M). Here we report that the flavin reductase FRase I from Vibrio fischeri is also a CB1954 nitroreductase, which has a substantially lower apparent K(M) than E. coli NfsB. To enhance the activity of FRase I with CB1954 we used targeted mutagenesis and an E. coli SOS reporter strain to engineer single- and multi-residue variants that possess a substantially reduced apparent K(M) and an increased k(cat)/K(M) relative to the wild type enzyme. In a bacteria-delivered model for enzyme prodrug therapy, the engineered FRase I variants were able to kill human colon carcinoma (HCT-116) cells at significantly lower CB1954 concentrations than wild type FRase I or E. coli NfsB.

Discovery and evaluation of Escherichia coli nitroreductases that activate the anti-cancer prodrug CB1954.

Gene-directed enzyme prodrug therapy (GDEPT) aims to achieve highly selective tumor-cell killing through the use of tumor-tropic gene delivery vectors coupled with systemic administration of otherwise inert prodrugs. Nitroaromatic prodrugs such as CB1954 hold promise for GDEPT as they are readily reduced to potent DNA alkylating agents by bacterial nitroreductase enzymes (NTRs). Transfection with the nfsB gene from Escherichia coli can increase the sensitivity of tumor cells to CB1954 by greater than 1000-fold. However, poor catalytic efficiency limits the activation of CB1954 by NfsB at clinically relevant doses. A lack of flexible, high-throughput screening technology has hindered efforts to discover superior NTR candidates. Here we demonstrate how the SOS chromotest and complementary screening technologies can be used to evaluate novel enzymes that activate CB1954 and other bioreductive and/or genotoxic prodrugs. We identify the major E. coli NTR, NfsA, as 10-fold more efficient than NfsB in activating CB1954 as purified protein (k(cat)/K(m)) and when over-expressed in an E. coli nfsA(-)/nfsB(-) gene deleted strain. NfsA also confers sensitivity to CB1954 when expressed in HCT-116 human colon carcinoma cells, with similar efficiency to NfsB. In addition, we identify two novel E. coli NTRs, AzoR and NemA, that have not previously been characterized in the context of nitroaromatic prodrug activation.

Characterization of PPTNs, a cyanobacterial phosphopantetheinyl transferase from Nodularia spumigena NSOR10.

The phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds, including fatty acids, polyketides, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by transfer of a phosphopantetheinyl moiety. The diverse PPT superfamily can be divided into two families based on specificity and conserved sequence motifs. The first family is typified by the Escherichia coli acyl carrier protein synthase (AcpS), which is involved in fatty acid synthesis. The prototype of the second family is the broad-substrate-range PPT Sfp, which is required for surfactin biosynthesis in Bacillus subtilis. Most cyanobacteria do not encode an AcpS-like PPT, and furthermore, some of their Sfp-like PPTs belong to a unique phylogenetic subgroup defined by the PPTs involved in heterocyst differentiation. Here, we describe the first functional characterization of a cyanobacterial PPT based on a structural analysis and subsequent functional analysis of the Nodularia spumigena NSOR10 PPT. Southern hybridizations suggested that this enzyme may be the only PPT encoded in the N. spumigena NSOR10 genome. Expression and enzyme characterization showed that this PPT was capable of modifying carrier proteins resulting from both heterocyst glycoplipid synthesis and nodularin toxin synthesis. Cyanobacteria are a unique and vast source of bioactive metabolites; therefore, an understanding of cyanobacterial PPTs is important in order to harness the biotechnological potential of cyanobacterial natural products.

The phosphopantetheinyl transferase superfamily: phylogenetic analysis and functional implications in cyanobacteria.

Phosphopantetheinyl transferases (PPTs) are a superfamily of essential enzymes required for the synthesis of a wide range of compounds including fatty acid, polyketide, and nonribosomal peptide metabolites. These enzymes activate carrier proteins in specific biosynthetic pathways by the transfer of a phosphopantetheinyl moiety to an invariant serine residue. PPTs display low levels of sequence similarity but can be classified into two major families based on several short motifs. The prototype of the first family is the broad-substrate-range PPT Sfp, which is required for biosynthesis of surfactin in Bacillus subtilis. The second family is typified by the Escherichia coli acyl carrier protein synthase (AcpS). Facilitated by the growing number of genome sequences available for analyses, large-scale phylogenetic studies were utilized in this research to reveal novel subfamily groupings, including two subfamilies within the Sfp-like family. In the present study degenerate oligonucleotide primers were designed for amplification of cyanobacterial PPT gene fragments. Subsequent phylogenetic analyses suggested a unique, function-based PPT type, defined by the PPTs involved in heterocyst differentiation. Evidence supporting this hypothesis was obtained by sequencing the region surrounding the partial Nodularia spumigena PPT gene. The ability to genetically classify PPT function is critical for the engineering of novel compounds utilizing combinatorial biosynthesis techniques. Information regarding cyanobacterial PPTs has important ramifications for the ex situ production of cyanobacterial natural products.