Publications
Peer-reviewed publications highlighting our recent work.
Priyanka Goyal, Dwight Deay III, Steve Seibold, A.C.L. Candido, Scott Lovell, Kevin P. Battaile, George S. Wilson, Mark L. Richter, and Peter A. Petillo
ABSTRACT: Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.
Dwight O. Deay III, Steve Seibold, Kevin P. Battaile, Scott Lovell, Mark L. Richter, and Peter A. Petillo
ABSTRACT: Demand exists for a nicotine oxidase enzyme with high catalytic efficiency for a variety of applications including the in vivo detection of nicotine, therapeutic enzymatic blockade of nicotine from the CNS, and inactivation of toxic industrial wastes generated in the manufacture of tobacco products. Nicotine oxidase enzymes identified to date suffer from low efficiency, exhibiting either a high kcat or low Km, but not both. Here we present the crystal structure of the (S)-6-hydroxy-nicotine oxidase from Shinella sp HZN7 (NctB), an enzyme that oxidizes (S)-nicotine with a high kcat (>1 s−1), that possesses remarkable structural and sequence similarity to an enzyme with a nanomolar Km for (S)-nicotine, the (S)-nicotine oxidase from Pseudomonas putidia strain S16 (NicA2). Based on a comparison of our NctB structure and the previously published crystal structure of NicA2, we successfully employed a rational design approach to increase the rate of oxidative turnover of the NicA2 enzyme by ∼25% (0.011 s−1 to 0.014 s−1), and reduce the Km of the NctB protein by approximately 34% (940 μM–622 μM). While modest, these results are a step towards engineering a nicotine oxidase with kinetic parameters that fulfill the functional requirements of biosensing, waste remediation, and therapeutic applications.
Pavel Yamanushkin, Sean P. Smith, Peter A. Petillo, and Michael Rubin
ABSTRACT: An expeditious click–click cyclize strategy for the assembly of medium-sized heterocyclic rings is described. The sequence involves the reaction of cycloprop-2-ene carboxylic acids with unsaturated amines to furnish amides, which are further subjected to a Cu-catalyzed directed carbomagnesiation and a ring-closing olefin metathesis reaction. This methodology allows for the efficient preparation of lactams with ring sizes up to 10.
Jonathon P. Matheny, Pavel M. Yamanushkin, Peter A. Petillo, and Michael Rubin
ABSTRACT: The sulfonamide moiety was evaluated as an activating and stabilizing functional group in the metal-templated strain release-driven intramolecular nucleophilic addition of amines to cyclopropenes to generate 1,5-diazocan-2-ones.
DOI: 10.1039/d0ra09014j
Dwight O. Deay III, Kim K. Colvert, Fei Gao, Steve Seibold, Priyanka Goyal, Daniel Aillon, Peter A. Petillo, and Mark L. Richter
ABSTRACT: The enzyme 6-Hydroxy-l-Nicotine oxidase (HLNO) is a flavin-dependent enzyme that catalyzes the first step in the pyridine pathway of oxidation of nicotine as a source of energy and nitrogen in several bacteria. Recombinant Arthrobacter nicotinovorans HLNO also catalyzes oxidation of (s)-nicotine at a low but measurable rate (Fitzpatrick et al., 2016, Biochemistry 55, 697–703). Rational design and bioinformatics approaches, based on the known high-resolution structure of this enzyme (RCSB: 3NG7), were employed to further enhance the catalytic turnover and stability of the enzyme using (S)-nicotine as substrate. The active site residue Tyr311 forms a hydrogen bond with the hydroxyl group of (S)-6-OH-nicotine within the catalytic pocket. Its replacement by a tryptophan residue reduced the kcat for (S)-6-OH-nicotine by more than 6-fold and increased ~1.5-fold. Combining this mutation with two surface mutations that were predicted to enhance enzyme stability, further increased the kcat for nicotine resulting in a comparatively robust oxidation of (s)-nicotine (kcat >1 s−1) at 37 °C, at the same time reducing the specificity for (S)–OH-nicotine (kcat/KM) by more than 100-fold and increasing that for (S)-nicotine by more than 2-fold. Interestingly, adding a maltose-binding protein (MBP) tag onto the N-terminus of HLNO markedly increased the thermal stability of the enzyme, extending the half-life at 37 °C from ~2 h to ~22 h. This effect was due almost entirely to increased FAD retention, an observation that may prove useful to improve flavin retention in other flavin-dependent monoamine oxidases.
Axel Scherer, Taeyoon Jeon, Changsoon Choi, Peter A Petillo, Jack Jewell, and John Richard Ordonez-Varela
ABSTRACT: A leak-detection system comprises networked imaging packages including an infrared camera based on microbolometer arrays, one or more filters, and a processor. The processor uses one or more novel processing methods for identifying gas leakages in a monitored facility. In some embodiments, the imaging packages are installed at various locations in the monitored facility. In some other embodiments, the leak-detection system is embodied as a mobile platform, wherein the networked imaging packages are coupled to drones that fly throughout the monitored facility to monitor for gas leakages. The flight path of the cameras may be pre-programmed and may further be alterable in real time. The novel processing technique(s) employed by the leak-detection system disclosed herein involve the use of multiple unique filters that facilitate enhancing the signal-to-noise ratio of captured images; that is, enhancing the contrast of a monitored gas against other objects. The novel processing techniques include one or more of the following: (i) two-image subtraction; (ii) time-resolved imaging; and (iii) multi-filter detection. Additionally, in some embodiments, the imaging packages include a visible (light) camera in addition to an IR camera.
Axel Scherer, Peter A Petillo, Samson Chen, and Azita Emami
ABSTRACT: A smart cage includes radiofrequency transceivers and tags attached to laboratory animals. The tags include sensors to detect monitorable conditions of the laboratory animals. The sensors include working electrodes, counter electrodes, reference electrodes, and potentiostats. The top surface of the electrodes is coated with ionophores or enzymes which detect the monitorable conditions of the laboratory animals.
Samson Chen, Axel Scherer, Dvin I. Adalian, Peter Petillo, Muhammad Musab Jilani, Xiomara L. Madero, and Deepan Kishore Kumar
ABSTRACT: A sensor implanted in tissues and including a sensing layer is fabricated by mixing the signal transduction enzyme with non-reactive components including buffer salts and fillers, and spin coating the enzyme onto a substrate. The signal transduction enzyme is crosslinked by introducing the coated substrate in a vacuum chamber. In the chamber, a crosslinker evaporates and is deposited onto the enzyme, therefore crosslinking the enzyme.
Axel A. Scherer, and Peter A. Petillo
ABSTRACT: A multilayer structure can selectively bind certain molecules, due to reentrant spaces having an appropriate size. The multilayers can be fabricated by alternating layers of two different materials having different etching rate. The layers of the material having a higher etching rate form reentrant spaces which can protect molecules from further chemical interactions.