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Studying the structure of an enzyme and how it relates to its function has been a goal of enzymologists for decades. Although informative and useful, classical techniques, such as BLAST searches or manual analysis of multisequence alignments may be too focused. These techniques tend to rely on comparing a manageable quantity of sequences. As a result, patterns in conservation of certain residues across a limited number of sequences carry more weight in the study. In the first section, we present a study that used these classical approaches to compare only 3 sequences of Aldolase Superfamily members as described below. NahE is a hydratase-aldolase that converts o-substituted trans-benzylidenepyruvates (where the ortho-substituent is H, OH, or CO₂⁻) to benzaldehyde, salicylaldehyde, or 2-carboxybenzaldehyde, respectively, and pyruvate. The enzyme is part of a bacterial pathway for the degradation of naphthalene, which is a toxic and persistent environmental contaminant. Sequence, crystallographic, and mutagenic analysis identified the enzyme as a member of the N-acetylneuraminate lyase (NAL) subgroup in the aldolase superfamily. As such, it has a conserved lysine (Lys183) and tyrosine (Tyr155), for Schiff base formation, as well as a GXXGE motif for binding of the pyruvoyl carboxylate group. NahE crystal structures show these core active site elements along with other nearby residues that might be involved in the mechanism and/or specificity. Mutations of five active site amino acids (Thr65, Trp128, Tyr155, Asn157, and Asn281) were constructed and kinetic parameters measured in order to assess the effect(s) on binding, catalysis, and/or the reaction step (hydration vs aldol cleavage). The results show that the two Trp128 mutants (Phe and Tyr) have the least effect on catalysis, whereas amino acids with bulky side chains at Thr65 (Val) and Asn281 (Leu) have the greatest effect. The Y155F mutation also significantly hinders catalysis and falls in between these extremes. These observations are put into a structural context. Finally, trapping experiments were carried out with substrate, NaCNBH₃, and wild type and selected mutations. The mass spectral analysis is consistent with the observed activities and suggests that pyruvate is released quickly from the active site, but salicylaldehyde is not. In the second section, we utilize a more modern technique of sequence analysis. We present studies done to analyze trends observed within the Tautomerase Superfamily (TSF) that have been identified by a sequence similarity network (SSN). Hidden trends in sequences that appear insignificant at the small scale may be revealed on a larger scale, such as in the second section. The amino-terminal proline (Pro1) has long been thought to be a mechanistic imperative for tautomerase superfamily (TSF) enzymes, functioning as a general base or acid in all characterized reactions. However, a global examination of more than 11,000 nonredundant sequences of the TSF uncovered 346 sequences that lack Pro1. The majority (~85%) are found in the malonate semialdehyde decarboxylase (MSAD) subgroup where most of the 294 sequences form a separate cluster. Four sequences within this cluster retain Pro1. Because these four sequences might provide clues to assist in the identification and characterization of activities of nearby sequences without Pro1, they were examined by kinetic, inhibition, and crystallographic studies. The most promising of the four (from Calothrix sp. PCC 6303 designated 437) exhibited decarboxylase and tautomerase activities and was covalently modified at Pro1 by 3-bromopropiolate. A crystal structure was obtained for the apo enzyme (2.35 Å resolution). The formation of a 3-oxopropanoate adduct with Pro1 provides clues to build a molecular model for the bound ligand. The modeled ligand extends into a region that allows interactions with three residues (Lys37, Arg56, Glu98), suggesting that these residues could play roles in the observed decarboxylation and tautomerization activities. Moreover, these same residues are conserved in 16 nearby, non-Pro1 sequences in a sequence similarity network. Thus far, these residues have not been implicated in the mechanisms of any other TSF members. The collected observations provide starting points for the characterization of the non-Pro1 sequences. Five non-Pro1 sequences were studied in this section, as well, containing either glycine, alanine, valine, threonine, or serine at the N-terminus. The most promising was NJ7 (from Nostoc sp. strain PCC 7120/SAG 25.82/UTEX 2576 derived from the UniProt Accession code Q8YNJ7). Kinetic analysis showed that this enzyme with Val1 has tautomerization and decarboxylation activity. The introduction of Pro1 enhanced NJ7’s performance as a tautomerase and decarboxylase.

Novel psychoactive substances (NPSs) are synthetically derived compounds designed to mimic the effects of other illicit drugs. An endless cycle of NPSs continually reach the drug market due to limitations in drug legislation creating two problems: (1) non-availability of standards and (2) multiple isobars indistinguishable even to high resolution mass spectrometry. Gas chromatography-mass spectrometry (GCMS) is the most common instrument used for compound identification in non-targeted seized drug analysis. Compound identification using this technique relies on mass spectra where the percent relative abundances (%RAs) of all m/z values are compared manually or searched against a library database. Lacking standards, laboratories rely on published GCMS spectra to postulate compound identification. The ability to make such identifications provides vital investigative data and may help uncover new structural variants. However, central to such identifications is an understanding of how much spectral patterns vary across laboratories; the assumption that variation is minimized through the use of a standard tuning compound perfluorotributylphosphine (PFTBA) has yet to be rigorously tested and demonstrated. The first focus of this research was the often-unappreciated aspect to any mass spectral comparison or search- the inherent variation in RAs. In the first study, the variation of mass spectral fragmentation patterns characterized by %RA was evaluated using three GCMS instruments and 16 NPS compounds. The variation of retention indices was also studied. Retention indices showed Decades ago, the Environmental Protection Agency (EPA) established tune check compounds as a means to control systematic variation across vendor platforms, which was the focus of the second study. The goal of the second study was to evaluate whether the decafluorotriphenylphosphine (DFTPP) criteria, as defined by the EPA, could be used to reduce the RA variation in mass spectra produced by NPSs. Instruments from two vendors were used to analyze 6 NPSs: 4 cannabinoids and 2 cathinones. Each NPS was analyzed 100 times per instrument; 10 replicates per tune repeated under 10 different tunes. Results showed that passing a DFTPP tune check was not correlated with reduced RA variation. Tuning algorithm differences between vendors did impact the %RA variance, but the frequency of instrument tuning was found to be the most critical factor for controlling RA variation. The results of this work suggest that forensic laboratories should develop quantitative metrics to evaluate autotuning results and define how these metrics will be used to dictate maintenance. This practice, coupled with tuning before each analytical batch, will reduce the variation of %RA values as much as practicable. Because GCMS usually cannot distinguish constitutional isomers, the second focus of this research was to develop a novel methodology for the NPS isomer discrimination. Ion mobility spectrometry-mass spectrometry (IMS-MS) is used as a non-confirmatory screening technique for illicit substances in high traffic areas. Through the years, advancements in instrumentation have improved detection limits, increased resolving power, and allowed for unique instrument modifications which improve structural inferences. Gas-phase hydrogen deuterium exchange (HDX) is one such modification technique. HDX-IMS-MS has been used primarily for proteomic and metabolomic applications, but it is also effective in discriminating of small biomolecules (glycan linkages). For the final study, NPSs containing labile heteroatom hydrogens were evaluated for HDX reactivity in the presence of either deuterated water (D2O) or ammonia (ND3) within the drift tube. An initial evaluation of exchange propensity was performed for six NPSs. Five of the initial NPSs exchanged in the presence of ND3, while only one NPS (benzyl piperazine) exchanged with D2O. The exchange mechanism of D2O requires stabilization with a nearby charged site; the diamine ring of benzyl piperazine provided this charge site at a fixed length. Three disubstituted benzene isomers (ortho-, meta-, and para-) containing the diamine ring structure and a fluorine atom were analyzed to determine if isomer discrimination was possible. These isomers had nearly identical collisional cross sections and isotopic distributions in the drift gas, therefore IMS and MS alone cannot discriminate between the isomers. A t-test of means (α=0.05) showed that discrimination was possible if the exchange data from both reagent gases were included. Molecular dynamics simulations showed that the proximity of the fluorine to the diamine ring hinders the dihedral angle between the benzene ring and the diamine ring. The angle limitation partially accounts the observed exchange differences. One clear limitation of this technique is that the substance must exchange in the presence of both reagent gases. Nevertheless, this technique was shown to be capable of isomer discrimination in non-targeted analyses of NPSs.