A covalent NAD intermediate in the urocanase reaction

Matherly, Larry H.; DeBrosse, Charles W.; Phillips, Allen T.

doi:10.1021/bi00540a033

Cited by 17 publications

(7 citation statements)

References 21 publications

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“…Most likely, the urocanase reaction runs through an intermediate forming a covalent bond between nicotinamide and the imidazole of urocanate. 18,19 Moreover, it has been established by deuteration experiments that the two H Re protons depicted in Figure 1 are derived from the solvent. 20 Here, we report the crystal structure of urocanase from P. putida, which reveals the sequestered urocanate position adjacent to the nicotinamide of NAD C and the residues participating in the catalysis.…”

Section: Introductionmentioning

confidence: 99%

Structure and Action of Urocanase

Kessler

Rétey

Schulz

2004

Journal of Molecular Biology

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Section: Introductionmentioning

confidence: 99%

Structure and Action of Urocanase

Kessler

Rétey

Schulz

2004

Journal of Molecular Biology

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“…25,26 In urocanase, a covalent intermediate between imidazolepropionate and NAD + exhibited an absorbance peak at 335 nm. 27 In that case, NMR showed that the imidazole nitrogen formed a covalent adduct at the C4 atom of the nicotinamide ring. The absorbance peak of 334 nm observed here for the covalent NAD−phosphine adduct is similar to that previously reported for various covalent NAD species.…”

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confidence: 99%

“…The nicotinamide ring of NAD + has electrophilic character and is susceptible to modifications by nucleophiles. Biologically relevant is the fact that sulfhydryl compounds such as cysteine and glutathione can form adducts at the C4 atom of the nicotinamide with absorbance maxima at 330–335 nm. , In urocanase, a covalent intermediate between imidazolepropionate and NAD + exhibited an absorbance peak at 335 nm . In that case, NMR showed that the imidazole nitrogen formed a covalent adduct at the C4 atom of the nicotinamide ring.…”

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Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD⁺-Dependent Enzymes

Patel

Smith

Morton³

et al. 2020

Biochemistry

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Protein biochemistry protocols typically include disulfide bond reducing agents to guard against unwanted thiol oxidation and protein aggregation. Commonly used disulfide bond reducing agents include dithiothreitol, β-mercaptoethanol, glutathione, and the tris(alkyl)phosphine compounds tris(2-carboxyethyl)phosphine (TCEP) and tris(3-hydroxypropyl)phosphine (THPP). While studying the catalytic activity of the NAD(P)H-dependent enzyme Δ 1 -pyrroline-5-carboxylate reductase, we unexpectedly observed a rapid non-enzymatic chemical reaction between NAD + and the reducing agents TCEP and THPP. The product of the reaction exhibits a maximum ultraviolet absorbance peak at 334 nm and forms with an apparent association rate constant of 231−491 M −1 s −1 . The reaction is reversible, and nuclear magnetic resonance characterization ( 1 H, 13 C, and 31 P) of the product revealed a covalent adduct between the phosphorus of the tris(alkyl)phosphine reducing agent and the C4 atom of the nicotinamide ring of NAD + . We also report a 1.45 Å resolution crystal structure of short-chain dehydrogenase/reductase with the NADP + −TCEP reaction product bound in the cofactor binding site, which shows that the adduct can potentially inhibit enzymes. These findings serve to caution researchers when using TCEP or THPP in experimental protocols with NAD(P) + . Because NAD(P) + -dependent oxidoreductases are widespread in nature, our results may be broadly relevant.

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“…Histidine ammonia-lyase, the initial enzyme in the pathway, contains dehydroalanine which functions in catalysis by an a,p-elimination mechanism which is not well understood (14). Urocanase, the second enzyme, contains one residue of tightly bound NAD whose role in catalysis appears to involve covalent attachment to the urocanate molecule rather than intermediate conversion to NADH (23). As part of our effort in studying these enzymes from Pseudomonas putida, we wished to examine their gene structure and control at the DNA level to obtain further information on the structural properties of both enzymes.…”

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Cloning and expression in Escherichia coli of histidine utilization genes from Pseudomonas putida

Consevage¹,

Porter²,

Phillips³

1985

J Bacteriol

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A library of the Psesdomonas putda chromosome, prepared through the use of the cosmid pJB8 ligated to a partial Sau3A digest of bacterial DNA, followed by in vitro pa ing into bacteriophage lambda particles, was used to construct a strain ofEscherichia coli which contained the genes for histidine utilization. This isolate produced a repressor product and all five enzymes required in Pseudomonas spp. for histidine dissimilation, whereas none of these could be detected in the nontransduced parent E. coli strain. When this transductant was grown on various media containing histidine or urocanate as the inducer, it was observed that production of the cloned histidine clegradative enzymes was influenced somewhat by the choice of nitrogen source used but not by the carbon source. The recombinant cosmid was isolated and found to consist of 21.1 kiolbase pairs of DNA, with approximately 16 kilobase pairs derived from Pseudomonas DNA and the remainder being from the pJB8 vector. Digestion of this insert DNA with EcoRI provided a 6.1-kilobase-pair fragment which, upon ligation in pUC8 and transformation into an E. coil host, was found to encode histidine ammonia-lyase and urocanase. The inducible nature of this production indicated that the hut repressor gene also was present on this fragment. Insertional inactivation of the histidine ammonia-lyase and urocanase genes by the gamma-delta transposon has permitted location of these structural genes and has provided evidence that transcription proceeds from urocanase through histidine ammonia-lyase. Mapping of the 16-kilobase-pair Pseudomonas DNA segment with restriction enzymes and subcloning of additional portions, one of which contained the gene for formiminoglutamate hydrolase and another that could constitutively express activities for both imidazolone propionate hydrolase and formylglutamate hydrolase, has provided evidence for the organization of all hut genes.

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A covalent NAD intermediate in the urocanase reaction

Cited by 17 publications

References 21 publications

Structure and Action of Urocanase

Structure and Action of Urocanase

Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD⁺-Dependent Enzymes

Cloning and expression in Escherichia coli of histidine utilization genes from Pseudomonas putida

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A covalent NAD intermediate in the urocanase reaction

Cited by 17 publications

References 21 publications

Structure and Action of Urocanase

Structure and Action of Urocanase

Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD+-Dependent Enzymes

Cloning and expression in Escherichia coli of histidine utilization genes from Pseudomonas putida

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Cautionary Tale of Using Tris(alkyl)phosphine Reducing Agents with NAD⁺-Dependent Enzymes