A. Genetic characterization of the caffeine C-8 oxidation pathway in Pseudomonas Sp. CBB1 B. Validation of caffeine dehydrogenase as a suitable enzyme for a rapid caffeine diagnostic test

Abstract

all of you have been instrumental in extending crucial technical help in a timely manner in my day-today research work. Dr. Yu was always around to help me in protein purification, characterization, gene cloning and all other microbial analysis techniques. Dr. Louie and Dr. Summers were always prompt in reciprocating to discussions concerned with this research of any related advanced molecular bio-techniques. Dr. Das and Dr. Gopishetty were active in providing inputs in terms of organic/analytical chemistry and structural analysis, including, but not limited to any technical questions pertaining to high-end instruments like LC-MS, NMR, etc. I will always be thankful to my previous co-workers, Bhavita Bhatt and Kaitlin Louie, for their diligent support in organizing and maintaining the daily chores of the lab which further facilitated me in conducting experiments without interruptions. And last, but not the least, Dr. Todor Genkov, whose charming discussions kept my spirits high and my mind productive through the toil of many workaday routines. I would like extend my sincere thanks to Dr. Santhana Velupillai, and Prof. Amnon Kohen and his student Eric Kohen in the Chemistry Department, for helping me with various experiments which were conducted in their respective laboratories. Our department secretaries-Linda Wheatley, Natalie Potter, Kay Spidle, Wendy Meyers-all deserve my deepest gratitude for their warm help within all student-related activities and their promptness in helping me in processing orders. Being an international student, it would have been impossible to even be functional in graduate school without their unwavering efforts in undergirding the university machinery. Last, but not the least, I would like to thank my parents, my teachers, and all my friends who have always extended their constant encouragement towards academic excellence. I will always be thankful to all of them and obliged, for their contribution in my academic success.

Genetic characterization of caffeine degradation by bacteria and its potential applications

“…Mohanty and colleagues () proposed a similar conversion of (R)‐(‐)‐TMA, formed by spontaneous degradation of TM‐HIU, to (S)‐(+)‐TMA by a TMA racemase. However, none of the genes in the 25.2‐kb CBB1 genetic cluster showed any homology to allantoin racemase (Mohanty, ). Thus, there yet remain undiscovered genes encoding enzymes active in the caffeine C‐8 oxidation pathway.…”

“…Two other genes in this cluster, tmuH and tmuD (Fig. inset), are proposed to encode putative enzymes (TM‐HIU hydroxylase and TM‐OHCU decarboxylase, respectively) of the C‐8 oxidation pathway (Mohanty et al ., ) based on their homology to enzymes of the uric acid metabolic pathway in K. pneumoniae (French and Ealick, 2010; 2011; Mohanty, ). The presence of these putative enzymes in CBB1 suggests enzymatic formation of S‐(+)‐TMA from TM‐HIU via TM‐OHCU in the C‐8 oxidation pathway (Mohanty et al ., ).…”

“…Recent work has revealed the nature of enzymes involved in both N-demethylation and C-8 oxidation pathways (Asano et al, 1994;Madyastha et al, 1999;Yamaoka-Yano and Mazzafera, 1999;Yu et al, 2008;2009;Summers et al, 2011;Mohanty, et al, 2012). The gene sequences for these enzymes have also been elucidated, reported and deposited in the GenBank database (Mohanty et al, 2012;Summers et al, 2012;2013). Currently, the only known bacterial genes responsible for caffeine metabolism are those that are directly responsible for catabolism.…”

“…Recently, the five enzymes (NdmABCDE) that catalyse the entire caffeine N-demethylation pathway in P. putida CBB5 were purified and characterized (Summers et al, 2011;2013). These same enzymes are also responsible for metabolism of theophylline in CBB5.…”

“…In addition, the gene sequences of all five enzymes (ndmABCDE) were determined from a 13.2-kb CBB5 genomic DNA fragment ( Fig. 1B) and deposited in the GenBank database (Summers et al, 2012;2013). NdmA and NdmB are Rieske [2Fe-2S] monooxygenases with a non-heme iron at the active site (Summers et al, 2012).…”