scholarly journals Absence of Substrate Channeling between Active Sites in theAgrobacterium tumefaciensIspDF and IspE Enzymes of the Methyl Erythritol Phosphate Pathway†

Biochemistry ◽  
2006 ◽  
Vol 45 (11) ◽  
pp. 3548-3553 ◽  
Author(s):  
Christian Lherbet ◽  
Florence Pojer ◽  
Stéphane B. Richard ◽  
Joseph P. Noel ◽  
C. D. Poulter
Keyword(s):  
Active Sites ◽  
Substrate Channeling ◽  
Methyl Erythritol Phosphate Pathway ◽  
Methyl Erythritol Phosphate

scholarly journals Structural and mutational analyses of the bifunctional arginine dihydrolase and ornithine cyclodeaminase AgrE from the cyanobacterium Anabaena

2020 ◽  
Vol 295 (17) ◽  
pp. 5751-5760
Author(s):  
Haehee Lee ◽  
Sangkee Rhee
Keyword(s):  
Binding Site ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Nitrogen Storage ◽  
Active Site Residues ◽  
Substrate Channeling ◽  
Arginine Dihydrolase ◽  
Arginine Catabolism ◽  
Cyanobacterium Anabaena ◽  
Ornithine Cyclodeaminase

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine–ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure–function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase–ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.

scholarly journals New flexibility/fold/interactions in the Pyruvate Dehydrogenase complex assembly

2014 ◽  
Vol 70 (a1) ◽  
pp. C427-C427
Author(s):  
William Furey
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Active Sites ◽  
Neurological Diseases ◽  
Pyruvate Dehydrogenase Complex ◽  
Control Point ◽  
Sugar Metabolism ◽  
Substrate Channeling ◽  
Simple Diffusion ◽  
Multiple Copies ◽  
Cellular Apoptosis

The pyruvate dehydrogenase multienzyme complex (PDHc), is a large, macromolecular machine that converts the product of glycolysis, pyruvate, to acetyl-coenzyme A, with the overall PDHc reaction functioning as a control point in carbohydrate metabolism. Altered levels of PDHc are associated with neurological diseases including Alzheimers and Parkinsons, and because of its role in sugar metabolism, its regulatory mechanisms are targets for controlling type 2 diabetes. Inhibition of PDHc's tissue specific, regulatory kinases increase mitochondrial levels of reactive oxygen species leading to cellular apoptosis and inhibition of tumor growth, suggesting a potential for treating cancer. Two major types of complexes are found: ~4.5 megadalton, octahedral complexes containing 60 enzymatic subunits and having a cubic core; and ~9 megadalton, icosahedral complexes containing ~120 enzymatic subunits and having a dodecahedral core. Multiple copies of at least 3 key enzymatic components, E1, E2 and E3 are always present, while the larger complexes may also contain regulatory phosphatases, kinases, and non-enzymatic, E2-like proteins. The assemblies and unusual substrate channeling mechanism employed by the complexes are fascinating, as intermediates are transferred between active sites by a long, highly flexible, "swinging arm" hand-delivery system employing lipoyl domains rather than by simple diffusion. We have crystallographically analyzed the enzymatic components of PDHc from E. coli, as well as some of their binary sub-complexes. We found that: key E1-E2 interactions stabilizing the overall assembly differ substantially (displaced by over 100 angstroms!) for complexes containing homodimeric vs heterotetrameric E1 components; that the conformation of parts of homodimeric E1's is dramatically stabilized in the presence of its E2 binding partner and reveals a new fold; and that models for octahedral PDHc complexes based on icosahedral complexes are likely to be incorrect.

A STUDY ON KINETIC CHARACTERIZATION OF CHANNEL-BLOCKING MUTANTS IN BRADYRHIZOBIUM JAPONICUM UTILIZATION A

2021 ◽  
pp. 49-52
Author(s):  
Anand Shanker Singh ◽  
G. Radhika ◽  
R. Praveen Kumar ◽  
Debarshi Jana
Keyword(s):  
Active Site ◽  
Bradyrhizobium Japonicum ◽  
Kinetic Data ◽  
Active Sites ◽  
Substrate Channeling ◽  
Site Analysis ◽  
Proline Utilization ◽  
Channel Blocking ◽  
Proline Utilization A

Proline utilization A (PutA) from Bradyrhizobium japonicum (BjPutA) is a bifunctional avoenzyme that catalyzes the oxidation of proline to glutamate using fused proline dehydrogenase (PRODH) and ∆1-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains. Recent crystal structures and kinetic data suggest an intramolecular channel connects the two active sites, promoting substrate channeling of the intermediate P5C from the PRODH domain to the P5CDH domain. In this work several mutations were made along the channel in an effort to block passage of P5C to the second active site. Analysis of several site-specic mutants in the substrate channel of BjPutA revealed an important role for D779 in the channeling path. BjPutA mutants D779Y and D779W signicantly decreased the overall PRODH-P5CDH channeling reaction indicating that bulky mutations at residue D779 impede travel of P5C through the channel. Interestingly, D779Y and D779W also exhibited lower P5CDH activity, suggesting that exogenous P5C must enter the channel upstream of D779. Replacing D779 with a smaller residue (D779A) had no effect on the catalytic and channeling properties of BjPutA showing that the carboxylate group of D779 is not essential for channeling. An identical mutation at D778 (D778Y) did not impact BjPutA channeling activity. Thus, D779 is optimally orientated so that replacement with the larger side chains of Tyr/Trp blocks P5C movment through the channel. The kinetic data reveal not only that bulky mutations at residue D779 hinder passage of P5C to the second active site, but also P5C must use the channel to efciently access the P5CDH domain. Moreover, these mutants may be used to learn more about the hydrolysis event that is thought to take place within the channel

scholarly journals Crystal structure of human mitochondrial trifunctional protein, a fatty acid β-oxidation metabolon

2019 ◽  
Vol 116 (13) ◽  
pp. 6069-6074 ◽  
Author(s):  
Chuanwu Xia ◽  
Zhuji Fu ◽  
Kevin P. Battaile ◽  
Jung-Ja P. Kim
Keyword(s):  
Crystal Structure ◽  
Active Sites ◽  
Acyl Chain ◽  
Protein A ◽  
Membrane Integrity ◽  
Fatty Acyl ◽  
Substrate Channeling ◽  
Mitochondrial Inner Membrane ◽  
Membrane Bound ◽  
Mitochondrial Trifunctional Protein

Membrane-bound mitochondrial trifunctional protein (TFP) catalyzes β-oxidation of long chain fatty acyl-CoAs, employing 2-enoyl-CoA hydratase (ECH), 3-hydroxyl-CoA dehydrogenase (HAD), and 3-ketothiolase (KT) activities consecutively. Inherited deficiency of TFP is a recessive genetic disease, manifesting in hypoketotic hypoglycemia, cardiomyopathy, and sudden death. We have determined the crystal structure of human TFP at 3.6-Å resolution. The biological unit of the protein is α2β2. The overall structure of the heterotetramer is the same as that observed by cryo-EM methods. The two β-subunits make a tightly bound homodimer at the center, and two α-subunits are bound to each side of the β2dimer, creating an arc, which binds on its concave side to the mitochondrial innermembrane. The catalytic residues in all three active sites are arranged similarly to those of the corresponding, soluble monofunctional enzymes. A structure-based, substrate channeling pathway from the ECH active site to the HAD and KT sites is proposed. The passage from the ECH site to the HAD site is similar to those found in the two bacterial TFPs. However, the passage from the HAD site to the KT site is unique in that the acyl-CoA intermediate can be transferred between the two sites by passing along the mitochondrial inner membrane using the hydrophobic nature of the acyl chain. The 3′-AMP-PPi moiety is guided by the positively charged residues located along the “ceiling” of the channel, suggesting that membrane integrity is an essential part of the channel and is required for the activity of the enzyme.

scholarly journals Structural basis of substrate channeling in the PutA flavoenzyme

2014 ◽  
Vol 70 (a1) ◽  
pp. C1162-C1162
Author(s):  
John Tanner
Keyword(s):  
Crystal Structures ◽  
Active Sites ◽  
Quaternary Structure ◽  
Structural Basis ◽  
Substrate Channeling ◽  
Oligomeric State ◽  
X Ray Crystallography ◽  
Proline Utilization ◽  
Domain Of Unknown Function ◽  
Saxs Analysis

Proline utilization A (PutA) is a high-hanging fruit of X-ray crystallography. PutA is a membrane-associated bifunctional flavoenzyme that catalyzes the 4-electron oxidation of proline to glutamate by the sequential activities of proline dehydrogenase and aldehyde dehydrogenase domains. PutAs are challenging crystallography targets because of their long polypeptide chain length (1000-1300 residues) and multidomain architecture. In this talk, I will present new crystal structures and SAXS analysis of two PutAs. Seven high resolution crystal structures of a 1004-residue minimalist PutA were determined using Hg SIRAS phasing, and the oligomeric state and quaternary structure were determined with SAXS [1]. The structures reveal an elaborate and dynamic tunnel system featuring a 75-Å long tunnel that links the two active sites. Also, a novel mechanism-based inactivation strategy allowed the trapping of the elusive PutA-quinone complex in the crystalline state. These structures provide insight into the mechanism of substrate channeling and how the enzyme changes conformation during the catalytic cycle. I will conclude by describing the first structure of a new type of PutA that contains an additional C-terminal domain of unknown function (CTDUF) that is not present in the smaller minimalist enzyme [2]. This larger PutA reveals an unexpectedly different structural solution to the problem of sequestering the reaction intermediate.

What catalysis needs from the electron microscopist

1988 ◽  
Vol 46 ◽  
pp. 694-695
Author(s):  
Alexis T. Bell
Keyword(s):  
Active Sites ◽  
Heterogeneous Catalysts ◽  
Catalyst Deactivation ◽  
Surface Composition ◽  
Internal Surface ◽  
Active Phase ◽  
Atomic Scale ◽  
Metal Catalyst ◽  
Internal Surface Area ◽  
Porous Support

Heterogeneous catalysts, used in industry for the production of fuels and chemicals, are microporous solids characterized by a high internal surface area. The catalyticly active sites may occur at the surface of the bulk solid or of small crystallites deposited on a porous support. An example of the former case would be a zeolite, and of the latter, a supported metal catalyst. Since the activity and selectivity of a catalyst are known to be a function of surface composition and structure, it is highly desirable to characterize catalyst surfaces with atomic scale resolution. Where the active phase is dispersed on a support, it is also important to know the dispersion of the deposited phase, as well as its structural and compositional uniformity, the latter characteristics being particularly important in the case of multicomponent catalysts. Knowledge of the pore size and shape is also important, since these can influence the transport of reactants and products through a catalyst and the dynamics of catalyst deactivation.

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