Presentation Abstract

Abstract Title: Purification, Cloning, and Functional Expression of NdmA and NdmB, Two Positional-Specific Methylxanthine N-Demethylases from Pseudomonas putida CBB5
Author Block: R. M. Summers, T. M. Louie, C. L. Yu, M. Subramanian;
Univ. of Iowa, Coralville, IA.
Presentation Number: 2794
Poster Board Number: 2794
Keywords: methylxanthine N-demethylase,caffeine
Abstract: Background: We have isolated a bacterial strain, Pseudomonas putida CBB5, capable of growing on caffeine as the sole carbon and nitrogen source. CBB5 degrades both caffeine and theophylline via sequential N-demethylation (NDM) to xanthine. We attempted to determine the number and nature of N-demethylase enzymes responsible for this metabolism.
Methods: Enzymes were purified by FPLC and biochemically characterized. N-demethylase genes were isolated from a genomic DNA library using a nested PCR approach and were cloned individually into the pET32a expression vector as C-terminal His-tagged fusion proteins. The expressed proteins were purified with a Ni-NTA column.
Results: A soluble, 2 subunit N-demethylase (Ndm) that degrades caffeine to xanthine was purified from CBB5. The two Ndm subunits, designated NdmA and NdmB, displayed apparent Mr of 40 and 35 kDa, respectively, and could not be resolved further. The NAD(P)H-dependent NDM reaction only occurs when Ndm couples with a partially purified reductase component and consumes one molecule of oxygen per methyl group removed as formaldehyde. Ndm was deduced as a Rieske [2Fe-2S] domain-containing non-heme iron oxygenase based on N-terminal sequence, UV absorbance spectrum, and iron content.
The gene sequences of both subunits were most similar to the catalytic subunits of other Riekse oxygenases known to cleave C-O and C-N bonds. Upon individual expression of the cloned Ndm genes, NdmA-His plus reductase N-demethylated caffeine to TB and was highly specific for the N-1 methyl group. In contrast, NdmB-His plus reductase was highly specific for N-3 methyl group, producing PX from caffeine. NdmA and B, which were not separable by wild-type purification, were fully resolved by the genetic approach.
Conclusions: Two positional-specific N-demethylases have been purified from P. putida CBB5. These enzymes have been cloned into and expressed by E. coli. To our knowledge, this is the first report of Rieske, non-heme iron oxygenases with position specific N-demethylase activity.