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Roles in binding and chemistry for conserved active site residues in the class 2 dihydroorotate dehydrogenase from Escherichia coli.

ABSTRACT: Dihydroorotate dehydrogenases (DHODs) catalyze the only redox step in de novo pyrimidine biosynthesis, the oxidation of dihydroorotate (DHO) to orotate (OA). During the reaction, the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of an enzyme-bound FMN prosthetic group as a hydride, and an active site base (Ser175 in the class 2 DHOD from Escherichia coli) deprotonates C5 of DHO. Aside from the identity of the active site base, the pyrimidine binding site of all DHODs is nearly identical. Several strictly conserved residues (four asparagines and either a serine or threonine) make extensive hydrogen bonds to the pyrimidine). The roles these conserved residues play in DHO oxidation are unknown. Site-directed mutagenesis was used to investigate the role of each residue during DHO oxidation. The effects of each mutation on substrate and product binding, as well as the effect on the rate constant of the chemical step, were determined. The effects of the mutations ranged from negligible to severe. Some of the residues were very important for chemistry, while others were important for binding. Mutation of residues capable of stabilizing reaction intermediates resulted in large decreases in the rate constant of the chemical step, suggesting these residues are quite important for stabilizing charge buildup in the active site. This finding is consistent with previous results that class 2 DHODs use a stepwise mechanism for DHO oxidation.

SUBMITTER: Fagan RL

PROVIDER: S-EPMC2800825 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Roles in binding and chemistry for conserved active site residues in the class 2 dihydroorotate dehydrogenase from Escherichia coli.

Fagan Rebecca L RL Palfey Bruce A BA

Biochemistry 20090801 30

Dihydroorotate dehydrogenases (DHODs) catalyze the only redox step in de novo pyrimidine biosynthesis, the oxidation of dihydroorotate (DHO) to orotate (OA). During the reaction, the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of an enzyme-bound FMN prosthetic group as a hydride, and an active site base (Ser175 in the class 2 DHOD from Escherichia coli) deprotonates C5 of DHO. Aside from the identity of the active site base, the pyrimidine binding site of all DHODs is ne ...[more]

PMID: 19530672

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Project description:Escherichia coli cysteine desulfurase (CD), IscS, modifies basal metabolism by transferring sulphur (S) from L-cysteine to numerous cellular pathways, whereas NFS1, a human CD, is active only in the formation of the [Acp]2:[ISD11]2:[NFS1]2 complex. Despite the accumulation of red-coloured IscS in E. coli cells as a result of the deficiency of accessible iron, as revealed in our previous studies, the mechanism of the potential enzymatic reaction remains unclear. In this study, the N-terminus of IscS was fused with the C-terminus of NFS1, which was reported to be almost fully active as IscS and exhibits a pyridoxal 5'-phosphate (PLP) absorption peak at 395 nm. Moreover, SUMO-EH-IscS exhibited significant growth recovery and NADH-dehydrogenase I activity in the iscS mutant cells. Furthermore, through in vitro and in vivo experiments combined with high-performance liquid chromatography and ultra-performance liquid chromatography-tandem mass spectrometry, it was shown that the new absorption peaks of the IscS H104Q, IscS Q183E, IscS K206A, and IscS K206A&C328S variants at 340 and 350 nm may correspond to the enzyme reaction intermediates, Cys-ketimine and Cys-aldimine, respectively. However, after mutation of the conserved active-site residues, additional absorption peaks at 420 and 430 nm were associated with PLP migration in the active-site pocket. Additionally, the corresponding absorption peaks of Cys-quinonoid, Ala-ketimine, and Ala-aldimine intermediates in IscS were 510, 325, and 345 nm, respectively, as determined by site-directed mutagenesis and substrate/product-binding analyses during the CD reaction process. Notably, red IscS formed in vitro by incubating IscS variants (Q183E and K206A) with excess L-alanine and sulphide under aerobic conditions produced an absorption peak similar to the wild-type IscS, at 510 nm. Interestingly, site-directed mutation of IscS with hydrogen bonds to PLP at Asp180 and Gln183 resulted in a loss of enzymatic activity followed by an absorption peak consistent with NFS1 (420 nm). Furthermore, mutations at Asp180 or Lys206 inhibited the reaction of IscS in vitro with L-cysteine (substrate) and L-alanine (product). These results suggest that the conserved active site residues (His104, Asp180, and Gln183) and their hydrogen bond with PLP in the N-terminus of IscS play a key role in determining whether the L-cysteine substrate can enter the active-site pocket and regulate the enzymatic reaction process. Therefore, our findings provide a framework for evaluating the roles of conserved active-site residues, motifs, and domains in CDs.

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Dataset Information

Roles in binding and chemistry for conserved active site residues in the class 2 dihydroorotate dehydrogenase from Escherichia coli.

Publications

Roles in binding and chemistry for conserved active site residues in the class 2 dihydroorotate dehydrogenase from Escherichia coli.

Similar Datasets

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