Bruce Allan Palfey

The emergence of respiratory, photosynthetic, and assimilatory complexes in evolution required proteins capable of binding multiple catalytic and electron-transfer cofactors while exerting fine control over their spatial arrangement. Across natural systems these cofactors are preferentially positioned in loop regions. In contrast, most protein design strategies have focused on installing cofactor-binding sites within helical elements. Here we show that introducing only a pair of appropriately placed histidine ligands into the interhelical loop regions of a canonical single-chain four-helix bundle is sufficient to create new well-defined high affinity heterocofactor binding sites. This simple modification enables the self-assembly of complexes containing up to three distinct cofactors in a single designed domain with positional specificity. Using this strategy, we creat-ed constructs containing one or two hemes in combination with Zn(II) phthalocyanine monosulfonate, Zn-heme, and the light-harvesting Zn(II) tetraphenylporphyrin tetrasulfonate. Fluorescence measurements of constructs containing the latter show efficient energy transfer between photoactive donor cofactors. By demonstrating that loop-embedded ligands support robust, modular, and evolutionarily plausible cofactor recruitment, this work provides a mechanistic explanation for the widespread placement of redox and catalytic cofactors in loops in natural proteins: only limited packing complementarity is needed, meaning that just a few mutations can introduce a functional cofactor binding site, after which additional mutations can tune affinity, reactivity, and specificity. More importantly, it establishes a straightforward path toward constructing func-tional protein domains that mirror the complexity of biological energy-conversion architectures.

The marine bacterial flavoenzymes Clz9 and Tcz9 can process cannabigerolic acid (CBGA) to the minor cannabinoid, cannabichromenic acid (CBCA), however, the mechanistic details of this extrinsic transformation are still obscure. Here, we report a thorough analysis of CBCA-formation by Clz9 and Tcz9 through high-resolution crystallographic characterization, biochemical analysis, and spectroscopic interrogation. Our work reveals that Clz9 and Tcz9 use different biochemical mechanisms from Cannabis cyclases and each other in their production of CBCA. Collection of a high-resolution substrate-bound structure, the first for any cannabinoid cyclase, provides key insights into how active site architecture affects substrate binding and stereoselectivity. Engineering approaches improve the stereoselectivity of CBCA formation by Clz9 and Tcz9, providing access to (R) and (S)-CBCA. Collectively, our work advances understanding of enzymatic cannabinoid formation and cements Clz9 and Tcz9 as two unique members of the BBE-like enzyme family with encouraging potential for biocatalytic cannabinoid production applications.

In Pseudomonas putida, the flavoprotein nicotine oxidoreductase (NicA2) catalyzes the oxidation of (S)-nicotine to N-methyl-myosmine, which is nonenzymatically hydrolyzed to pseudooxynicotine. Structural analysis reveals a monoamine oxidase (MAO)-like fold with a conserved FAD-binding domain and variable substrate-binding domain. The flavoenzyme has a unique variation of the classic aromatic cage with flanking residue pair W427/N462. Previous mechanistic studies using O₂ as the oxidizing substrate show that NicA2 has a low apparent K_m of 114 nM for (S)-nicotine with a very low apparent turnover number (k_cat of 0.006 s^–1). Herein, the mechanism of NicA2 was analyzed by transient kinetics. Single-site variants of W427 and N462 were used to probe the roles of these residues. Although several variants had moderately higher oxidase activity (7–12-fold), their reductive half-reactions using (S)-nicotine were generally significantly slower than that of wild-type NicA2. Notably, the reductive half-reaction of wild-type NicA2 is 5 orders of magnitude faster than the oxidative half-reaction with an apparent pseudo-first-order rate constant for the reaction of oxygen similar to k_cat. X-ray crystal structures of the N462V and N462Y/W427Y variants complexed with (S)-nicotine (at 2.7 and 2.3 Å resolution, respectively) revealed no significant active-site rearrangements. A second substrate-binding site was identified in N462Y/W427Y, consistent with observed substrate inhibition. Together, these findings elucidate the mechanism of a flavoenzyme that preferentially oxidizes tertiary amines with an efficient reductive half-reaction and a very slow oxidative half-reaction when O₂ is the oxidizing substrate, suggesting that the true oxidizing agent is unknown.

Ferulic acid decarboxylase catalyzes the decarboxylation of various substituted phenylacrylic acids to their corresponding styrene derivatives and CO₂ using the recently discovered cofactor prenylated FMN (prFMN). The mechanism involves an unusual 1,3-dipolar cycloaddition reaction between prFMN and the substrate to generate a cycloadduct capable of undergoing decarboxylation. Using native mass spectrometry, we show the enzyme forms a stable prFMN–styrene cycloadduct that accumulates on the enzyme during turnover. Pre-steady state kinetic analysis of the reaction using ultraviolet–visible stopped-flow spectroscopy reveals a complex pattern of kinetic behavior, best described by a half-of-sites model involving negative cooperativity between the two subunits of the dimeric enzyme. For the reactive site, the cycloadduct of prFMN with phenylacylic acid is formed with a k_app of 131 s^–1. This intermediate converts to the prFMN–styrene cycloadduct with a k_app of 75 s^–1. Cycloelimination of the prFMN–styrene cycloadduct to generate styrene and free enzyme appears to determine k_cat for the overall reaction, which is 11.3 s^–1.

Doxorubicin is an anthracycline chemotherapeutic produced by Streptomyces peucetius . It is an effective chemotherapeutic but has severe side effects and high toxicity, and the details of its biosynthetic pathway are largely unknown. Baumycin is a further decorated form of doxorubicin with an additional seven‐carbon acetal moiety attached to the anthracycline backbone of the molecule. In this work, we report our progress on the characterization of DnmZ, a Class D flavin monooxygenase involved in formation of the baumycin acetal. DnmZ is a N‐oxygenase that oxidizes an sugar‐linked exocyclic amine, triggering a nonenzymatic retro oxime‐aldol cleavage of the hexose. We aim to characterize the kinetics of the DnmZ‐catalyzed reaction between flavin and oxygen using stopped flow spectrophotometry. Results from this research can aid in the development of derivatized anthracyclines and the understanding of the class of enzymes known as the class D flavin monooxygenases. Our transient‐state experiments confirm that DnmZ catalyzes the formation of the C4a‐(hydro)peroxyflavin intermediate (C4a‐OOH). We have identified the appropriate wavelengths to monitor to observe C4a‐OOH formation (378nm) and elimination (450 and 480 nm). With this information we have quantitated flavin binding and determined that DnmZ’s binding affinity for flavin is approximately 13 μM, which compares well with other Class D monooxygenases. We are also investigating the effect of pH (pH 5.5 to 10.5) on C4a‐OOH formation and elimination. Our preliminary results suggest that increasing pH speeds up C4a‐OOH elimination, but has no effect on the intermediate’s formation. Support or Funding Information: California State University Program for Education & Research in BiotechnologyNIH SCORE 5SC3GM122652

Heteroaromatic sulfones react with cysteine via nucleophilic aromatic substitution, providing a mechanistically selective and irreversible scaffold for cysteine conjugation. Here we evaluate a library of heteroaromatic sulfides with different oxidation states, heteroatom substitutions, and a series of electron donating and electron-withdrawing substituents. Select substitutions profoundly influence reactivity and stability compared to conventional cysteine conjugation reagents, increasing the reaction rate by >3-orders of magnitude. The findings establish a series of synthetically accessible electrophilic scaffolds tunable across multiple tunable centers. New electrophiles and their corresponding alkyne-conjugates were profiled directly in cul-tured cells, achieving thiol saturation in a few minutes at sub-millimolar concentrations. Direct addition of desthiobiotin-functionalized probes to cultured cells simplified enrichment and elution to enable mass spectrometry discovery of >3000 reactive and/or accessible thiols labeled in their native cellular environments in a fraction of the standard analysis time. Surprisingly, only 1/2 of annotated cysteines were identified by both iodoacetamide-desthiobiotin and methylsulfonylbenzothiazole-desthiobiotin in replicate experiments, demonstrating complementary detection by mass spectrometry analysis. These probes offer advantages over existing cysteine alkylation reagents, including accelerated reaction rates, improved stability, and robust ionization for mass spectrometry applications. Overall, heteroaromatic sulfones provide modular tunability, shifted chromatographic elution times, and superior in-cell cysteine profiling for in-depth proteome-wide analysis and covalent ligand discovery.

Biocatalytic reactions embody many features of ideal chemical transformations, including the potential for impeccable selectivity, high catalytic efficiency, mild reaction conditions, and the use of environmentally benign reagents. These advantages have created a demand for biocatalysts that expand the portfolio of complexity-generating reactions available to synthetic chemists. However, the trade-off that often exists between the substrate scope of a biocatalyst and its selectivity limits the application of enzymes in synthesis. We recently demonstrated that a flavin-dependent monooxygenase, TropB, maintains high levels of site- and stereoselectivity across a range of structurally diverse substrates. Herein, we disclose the structural basis for substrate binding in TropB, which performs a synthetically challenging asymmetric oxidative dearomatization reaction with exquisite site- and stereoselectivity across a range of phenol substrates, providing a foundation for future protein engineering and reaction development efforts. Our hypothesis for substrate binding is informed by a crystal structure of TropB and molecular dynamics simulations with the corresponding computational TropB model and is supported by experimental data. In contrast to canonical class A FAD-dependent monooxygenases in which substrates bind in a protonated form, our data indicate that the phenolate form of the substrate binds in the active site. Furthermore, the substrate position is controlled through two-point binding of the phenolate oxygen to Arg206 and Tyr239, which are shown to have distinct and essential roles in catalysis. Arg206 is involved in the reduction of the flavin cofactor, suggesting a role in flavin dynamics. Further, QM/MM simulations reveal the interactions that govern the facial selectivity that leads to a highly enantioselective transformation. Thus, the structural origins of the high levels of site- and stereoselectivity observed in reactions of TropB across a range of substrates are elucidated, providing a foundation for future protein engineering and reaction development efforts.

In this contribution, the entangled two-photon absorption (ETPA) process on naturally occurring flavoproteins was studied. Low temperature responsive protein (LOT6P) and b-Type dihydroorotate dehydrogenase (DHODB) which possess flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) chromophores embedded in the protein environment were investigated. ETPA cross-section was measured and we found that increases when going from aqueous solution of the free flavin chromophore to the chromophore embedded in the protein. This enhancement is particularly evident when entangled photons are used as excitation light compared to classical light. Our results prove the potential of ETPA as a sensing technique for fluorescent proteins even for those whose classical TPA cross-section is small compared to well-known fluorescent proteins.