Characterization of the dinuclear metal center of <i>Pyrococcus furiosus</i> prolidase by analysis of targeted mutants

Du, Xuelian; Tove, Sherry R.; Kast-Hutcheson, Karen; Grunden, Amy M.

doi:10.1016/j.febslet.2005.09.086

Cited by 29 publications

(17 citation statements)

References 36 publications

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“…In hRecProl-G448R the kinetic parameters were indicative of a substantial active site structural perturbation, while the results on the hRecProl-231delY suggested that the deletion may perturb the substrate positioning/access to the catalytic site with a consequent decrease in the rate of catalysis. For the hRecProl-E412K, a higher K M value indicated that, besides being a ligand of the metal [38], residue 412 may also be involved in substrate binding/access.…”

Section: Discussionmentioning

confidence: 99%

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

et al. 2013

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Prolidase is the only human enzyme responsible for the digestion of iminodipeptides containing proline or hydroxyproline at their C-terminal end, being a key player in extracellular matrix remodeling. Prolidase deficiency (PD) is an intractable loss of function disease, characterized by mutations in the prolidase gene. The exact causes of activity impairment in mutant prolidase are still unknown. We generated three recombinant prolidase forms, hRecProl-231delY, hRecProl-E412K and hRecProl-G448R, reproducing three mutations identified in homozygous PD patients. The enzymes showed very low catalytic efficiency, thermal instability and changes in protein conformation. No variation of Mn(II) cofactor affinity was detected for hRecProl-E412K; a compromised ability to bind the cofactor was found in hRecProl-231delY and Mn(II) was totally absent in hRecProl-G448R. Furthermore, local structure perturbations for all three mutants were predicted by in silico analysis. Our biochemical investigation of the three causative alleles identified in perturbed folding/instability, and in consequent partial prolidase degradation, the main reasons for enzyme inactivity. Based on the above considerations we were able to rescue part of the prolidase activity in patients’ fibroblasts through the induction of Heath Shock Proteins expression, hinting at new promising avenues for PD treatment.

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Section: Discussionmentioning

confidence: 99%

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

et al. 2013

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“…The fact that cobalt ion can often substitute for zinc in vitro, giving comparable or higher levels of enzyme activity, is known (14,15) and suggests that cobalt and zinc can bind to similar motifs. In fact, Glu, Asp, and His were shown to coordinate Co 2ϩ in the cobalt-dependent prolidase of Pyrococcus furiosus (7,10).…”

Section: Discussionmentioning

confidence: 99%

Gulosibacter molinativorax ON4 ^T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase

et al. 2011

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A new pathway of molinate mineralization has recently been described. Among the five members of the mixed culture able to promote such a process, Gulosibacter molinativorax ON4 T has been observed to promote the initial breakdown of the herbicide into ethanethiol and azepane-1-carboxylate. In the current study, the gene encoding the enzyme responsible for molinate hydrolysis was identified and heterologously expressed, and the resultant active protein was purified and characterized. Nucleotide sequence analysis revealed that the gene encodes a 465-amino-acid protein of the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily with a predicted molecular mass of 50.9 kDa. Molinate hydrolase shares the highest amino acid sequence identity (48 to 50%) with phenylurea hydrolases of Arthrobacter globiformis and Mycobacterium brisbanense. However, in contrast to previously described members of the metal-dependent hydrolase A subfamily, molinate hydrolase contains cobalt as the only active-site metal.Molinate is a herbicide which is extensively applied to rice fields worldwide. When this thiocarbamate herbicide is applied to the flooded paddies, it dissipates into the environment largely through volatilization. However, (photo)chemical and microbiological molinate transformation also occurs (28), resulting in accumulation of oxidized metabolites, such as oxomolinate and molinate sulfoxide (11, 13), which have increased toxicity (5). The only biological system described so far as being able to mineralize molinate and use the herbicide as the sole source of carbon, energy, and nitrogen is a five-membered bacterial mixed culture (2, 6). Among the five community members, Gulosibacter molinativorax ON4 T (gen. nov., sp. nov.), the only representative of this genus thus far (21), is responsible for the initial breakdown of molinate. In contrast to previously described molinate transformation reactions (13), G. molinativorax ON4T cleaves the thioester bond of molinate, releasing ethanethiol and azepane-1-carboxylate (ACA) (Fig. 1) (1). While this hydrolysis proceeds in the absence of oxygen, the further metabolism and mineralization of ACA by G. molinativorax ON4 T necessitate oxygen. Ethanethiol is not transformed by this organism but is spontaneously oxidized to diethyl disulfide. At molinate concentrations of Ͼ2 mM, the accumulating sulfur compounds are toxic for G. molinativorax ON4T and molinate mineralization is achieved only when other mixed-culture members able to degrade the sulfurcontaining metabolites are present (1, 2).In the present study, the molinate hydrolase (MolA) from G. molinativorax ON4T responsible for the key step of molinate breakdown was purified and characterized. The encoding gene was identified and expressed in Escherichia coli. This enzyme belongs to the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily; however, in contrast to previously characterized members, it contains cobalt as a cofactor. To the best of our knowledge, this is the first description of an...

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“…In bacteria and archaea, prolidase is thought to function in the degradation of intracellular proteins and in proline recycling in concert with other endo‐ and exopeptidases (Du et al. 2005).…”

Section: Introductionmentioning

confidence: 99%

Prolidase function in proline metabolism and its medical and biotechnological applications

Kitchener

Grunden

2012

J Appl Microbiol

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Summary Prolidase is a multifunctional enzyme that possesses the unique ability to degrade imidodipeptides in which a proline or hydroxyproline residue is located at the C‐terminal end. Prolidases have been isolated from archaea and bacteria, where they are thought to participate in proline recycling. In mammalian species, prolidases are found in the cytoplasm and function primarily to liberate proline in the final stage of protein catabolism, particularly during the biosynthesis and degradation of collagen. Collagen comprises nearly one‐third of the total protein in the body, and it is essential in maintaining tissue structure and integrity. Prolidase deficiency (PD), a rare autosomal recessive disorder in which mutations in the PEPD gene affect prolidase functionality, tends to have serious and sometimes life‐threatening clinical symptoms. Recombinant prolidases have many applications and have been investigated not only as a possible treatment for PD, but also as a part of anti‐cancer strategies, a component of biodecontamination cocktails and in the dairy industry. This review will serve to discuss the many in vivo functions of procaryotic and eucaryotic prolidases, as well as the most recent advances in therapeutic and biotechnological application of prolidases.

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Characterization of the dinuclear metal center of Pyrococcus furiosus prolidase by analysis of targeted mutants

Cited by 29 publications

References 36 publications

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

Gulosibacter molinativorax ON4 ^T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase

Prolidase function in proline metabolism and its medical and biotechnological applications

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Characterization of the dinuclear metal center of Pyrococcus furiosus prolidase by analysis of targeted mutants

Cited by 29 publications

References 36 publications

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

Kinetic and Structural Evidences on Human Prolidase Pathological Mutants Suggest Strategies for Enzyme Functional Rescue

Gulosibacter molinativorax ON4 T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase

Prolidase function in proline metabolism and its medical and biotechnological applications

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Gulosibacter molinativorax ON4 ^T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase