Characterization of Glycerol Trinitrate Reductase (NerA) and the Catalytic Role of Active-Site Residues

Abstract

Glycerol trinitrate reductase (NerA) from Agrobacterium radiobacter, a member of the old yellow enzyme (OYE) family of oxidoreductases, was expressed in and purified from Escherichia coli. Denaturation of pure enzyme liberated flavin mononucleotide (FMN), and spectra of NerA during reduction and reoxidation confirmed its catalytic involvement. Binding of FMN to apoenzyme to form the holoenzyme occurred with a dissociation constant of ca. 10 ؊7 M and with restoration of activity. The NerA-dependent reduction of glycerol trinitrate (GTN; nitroglycerin) by NADH followed ping-pong kinetics. A structural model of NerA based on the known coordinates of OYE showed that His-178, Asn-181, and Tyr-183 were close to FMN in the active site. The NerA mutation H178A produced mutant protein with bound FMN but no activity toward GTN. The N181A mutation produced protein that did not bind FMN and was isolated in partly degraded form. The mutation Y183F produced active protein with the same k cat as that of wild-type enzyme but with altered K m values for GTN and NADH, indicating a role for this residue in substrate binding. Correlation of the ratio of K m GTN to K m NAD(P)H , with sequence differences for NerA and several other members of the OYE family of oxidoreductases that reduce GTN, indicated that Asn-181 and a second Asn-238 that lies close to Tyr-183 in the NerA model structure may influence substrate specificity.

Growth of Arthrobacter sp. Strain JBH1 on Nitroglycerin as the Sole Source of Carbon and Nitrogen

“…Because all nitrogen was accounted for throughout, we conclude that the only nitrogen-containing intermediate compounds are 1,2DNG and 1MNG, which is consistent with previous studies (6,9,20). The fact that most of the nitrogen was released as nitrite is consistent with previous reports of denitration catalyzed by reductase enzymes (4,8,21). The minor amounts of nitrate observed could be from abiotic hydrolysis (5,12) or from oxidation of nitrite.…”

“…Pure cultures capable of completely denitrating NG as a source of nitrogen when provided additional sources of carbon include Bacillus thuringiensis/cereus and Enterobacter agglomerans (11) and a Rhodococcus species (8,9). Cultures capable of incomplete denitration to MNG in the presence of additional carbon sources were identified as Pseudomonas putida, Pseudomonas fluorescens (4), an Arthobacter species, a Klebsiella species (8,9), and Agrobacterium radiobacter (20).…”

“…The use of NG as a source of nitrogen has been studied in mixed and pure cultures during growth on alternative sources of carbon and energy (3,9,11,20). Under such conditions, NG undergoes a sequential denitration pathway in which NG is transformed to 1,2-dinitroglycerin (1,2DNG) or 1,3DNG followed by 1-mononitroglycerin (1MNG) or 2MNG and then glycerol, under both aerobic and anaerobic conditions (3,6,9,11,20), and the enzymes involved in denitration have been characterized in some detail (4,8,15,21). Pure cultures capable of completely denitrating NG as a source of nitrogen when provided additional sources of carbon include Bacillus thuringiensis/cereus and Enterobacter agglomerans (11) and a Rhodococcus species (8,9).…”

“…Under such conditions, NG undergoes a sequential denitration pathway in which NG is transformed to 1,2-dinitroglycerin (1,2DNG) or 1,3DNG followed by 1-mononitroglycerin (1MNG) or 2MNG and then glycerol, under both aerobic and anaerobic conditions (3,6,9,11,20), and the enzymes involved in denitration have been characterized in some detail (4,8,15,21). Pure cultures capable of completely denitrating NG as a source of nitrogen when provided additional sources of carbon include Bacillus thuringiensis/cereus and Enterobacter agglomerans (11) and a Rhodococcus species (8,9). Cultures capable of incomplete denitration to MNG in the presence of additional carbon sources were identified as Pseudomonas putida, Pseudomonas fluorescens (4), an Arthobacter species, a Klebsiella species (8,9), and Agrobacterium radiobacter (20).…”

Key Enzymes Enabling the Growth of Arthrobacter sp. Strain JBH1 with Nitroglycerin as the Sole Source of Carbon and Nitrogen

“…(14), and xenobiotic reductases (XenA and XenB) from Pseudomonas putida and Pseudomonas fluorescens (6). All of the enzymes are members of the old yellow enzyme (OYE) family (23, 33), require flavin mononucleotide (FMN), use NADPH as an electron donor, and catalyze the reductive elimination of nitrate ester groups from NG with concomitant release of nitrite (7,15,23,33). In all cases, both 1,2-and 1,3-DNG are produced, although selectivity for attack at either C1 or C2 has been observed in some cases (7,28).…”