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The tautomerase superfamily (TSF) is a collection of enzymes and proteins that share a simple β-α-β structural scaffold. Most members are constructed from a single-core β-α-β motif or two consecutively fused β-α-β motifs in which the N-terminal proline (Pro-1) plays a key and unusual role as a catalytic residue. The cumulative evidence suggests that a gene fusion event took place in the evolution of the TSF followed by duplication (of the newly fused gene) to result in the diversification of activity that is seen today. Analysis of the sequence similarity network (SSN) for the TSF identified several linking proteins ("linkers") whose similarity links subgroups of these contemporary proteins that might hold clues about structure-function relationship changes accompanying the emergence of new activities. A previously uncharacterized pair of linkers (designated N1 and N2) was identified in the SSN that connected the 4-oxalocrotonate tautomerase (4-OT) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) subgroups. N1, in the cis-CaaD subgroup, has the full complement of active site residues for cis-CaaD activity, whereas N2, in the 4-OT subgroup, lacks a key arginine (Arg-39) for canonical 4-OT activity. Kinetic characterization and nuclear magnetic resonance analysis show that N1 has activities observed for other characterized members of the cis-CaaD subgroup with varying degrees of efficiencies. N2 is a modest 4-OT but shows enhanced hydratase activity using allene and acetylene compounds, which might be due to the presence of Arg-8 along with Arg-11. Crystallographic analysis provides a structural context for these observations.

Respiratory syncytial virus (RSV) infection is the most common cause of infant hospitalizations worldwide, and in the absence of an RSV vaccine, antibody prophylaxis is the only means of protection. The two types of RSV, RSVA and RSVB, share a high level of amino acid identity but they have different serum neutralization profiles. Here, we describe a single amino acid at position 305 in the RSV fusion glycoprotein (RSV-F) that switches the profile from RSVA (Leucine-L) to RSVB (Isoleucine-I). We found that the L305I mutation caused protein-wide shape-shift, altering both antibody and patient serum neutralization susceptibility. We show that viral replication in the presence of selective pressure forces mutation at position 305 and was the only amino acid mutation to occur in RSVA at evolutionary bottlenecks. We also identified a new mutation in RSVB that was associated with pressure from antibody neutralization. We engineered the L305I mutation in an RSV reverse genetic model system that led to a switch in susceptibility between RSVA and B patient sera. We postulate that this event was a significant determinant in the divergence of RSVA and RSVB from the parent virus, and a new mutation in RSVB suggests these viruses are still diverging. Our work suggests that the most effective sterilizing antiviral immunity leads to more rapid virus mutation and immune evasion that applies to RSV treatment, vaccines, and surveillance.

Summary Polycyclic aromatic hydrocarbons (PAHs) are highly toxic, pervasive environmental pollutants with mutagenic, teratogenic, and carcinogenic properties. There is interest in exploiting the nutritional capabilities of microbes to remove PAHs from various environments including those impacted by improper disposal or spills. Although there is a considerable body of literature on PAH degradation, the substrates and products for many of the enzymes have never been identified and many proposed activities have never been confirmed. This is particularly true for high molecular weight PAHs (e.g., phenanthrene, fluoranthene, and pyrene). As a result, pathways for the degradation of these compounds are proposed to follow one elucidated for naphthalene with limited experimental verification. In this pathway, ring fission produces a species that can undergo a non-enzymatic cyclization reaction. An isomerase opens the ring and catalyzes a cis to trans double bond isomerization. The resulting product is the substrate for a hydratase-aldolase, which catalyzes the addition of water to the double bond of an α,β-unsaturated ketone, followed by a retro-aldol cleavage. Initial kinetic and mechanistic studies of the hydratase-aldolase in the naphthalene pathway (designated NahE) and two hydratase-aldolases in the phenanthrene pathway (PhdG and PhdJ) have been completed. Crystallographic work on two of the enzymes (NahE and PhdJ) provides a rudimentary picture of the mechanism and a platform for future work to identify the structural basis for catalysis and the individual specificities of these hydratase-aldolases.

NahE and PhdJ are bifunctional hydratase-aldolases in bacterial catabolic pathways for naphthalene and phenanthrene, respectively. Bacterial species with these pathways can use polycyclic aromatic hydrocarbons (PAHs) as sole sources of carbon and energy. Because of the harmful properties of PAHs and their widespread distribution and persistence in the environment, there is great interest in understanding these degradative pathways, including the mechanisms and specificities of the enzymes found in the pathways. This knowledge can be used to develop and optimize bioremediation techniques. Although hydratase-aldolases catalyze a major step in the PAH degradative pathways, their mechanisms are poorly understood. Sequence analysis identified NahE and PhdJ as members of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. Both have a conserved lysine and tyrosine (for Schiff base formation) as well as a GXXGE motif (to bind the pyruvoyl carboxylate group). Herein, we report the structures of NahE, PhdJ, and PhdJ covalently bound to substrate via a Schiff base. Structural analysis and dynamic light scattering experiments show that both enzymes are tetramers. A hydrophobic helix insert, present in the active sites of NahE and PhdJ, might differentiate them from other NAL subgroup members. The individual specificities of NahE and PhdJ are governed by Asn-281/Glu-285 and Ser-278/Asp-282, respectively. Finally, the PhdJ complex structure suggests a potential mechanism for hydration of substrate and subsequent retro-aldol fission. The combined findings fill a gap in our mechanistic understanding of these enzymes and their place in the NAL subgroup.