All the O2 Consumed by Thermus thermophilus Cytochrome ba3 Is Delivered to the Active Site through a Long, Open Hydrophobic Tunnel with Entrances within the Lipid Bilayer

Fatty-acid degradation is an oxidative process that involves four enzymatic steps and is referred to as the β-oxidation pathway. During this process, long-chain acyl-CoAs are broken down into acetyl-CoA, which enters the mitochondrial tricarboxylic acid (TCA) cycle, resulting in the production of energy in the form of ATP. Enoyl-CoA hydratase (ECH) catalyzes the second step of the β-oxidation pathway by the syn addition of water to the double bond between C2 and C3 of a 2-trans-enoyl-CoA, resulting in the formation of a 3-hydroxyacyl CoA. Here, the crystal structure of ECH from Thermus thermophilus HB8 (TtECH) is reported at 2.85 Å resolution. TtECH forms a hexamer as a dimer of trimers, and wide clefts are uniquely formed between the two trimers. Although the overall structure of TtECH is similar to that of a hexameric ECH from Rattus norvegicus (RnECH), there is a significant shift in the positions of the helices and loops around the active-site region, which includes the replacement of a longer α3 helix with a shorter α-helix and 310-helix in RnECH. Additionally, one of the catalytic residues of RnECH, Glu144 (numbering based on the RnECH enzyme), is replaced by a glycine in TtECH, while the other catalytic residue Glu164, as well as Ala98 and Gly141 that stabilize the enolate intermediate, is conserved. Their putative ligand-binding sites and active-site residue compositions are dissimilar.

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Molecular basis for metabolite channeling in a ring opening enzyme of the phenylacetate degradation pathway

Nature Communications ◽

10.1038/s41467-019-11931-1 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Nitish Sathyanarayanan ◽

Giuseppe Cannone ◽

Lokesh Gakhar ◽

Nainesh Katagihallimath ◽

Ramanathan Sowdhamini ◽

...

Keyword(s):

Conformational Changes ◽

Active Site ◽

Ring Opening ◽

Environmental Pollutants ◽

Degradation Pathway ◽

Substrate Channeling ◽

Enzyme Form ◽

X Ray Scattering ◽

Hydrophobic Tunnel ◽

Metabolite Channeling

Abstract Substrate channeling is a mechanism for the internal transfer of hydrophobic, unstable or toxic intermediates from the active site of one enzyme to another. Such transfer has previously been described to be mediated by a hydrophobic tunnel, the use of electrostatic highways or pivoting and by conformational changes. The enzyme PaaZ is used by many bacteria to degrade environmental pollutants. PaaZ is a bifunctional enzyme that catalyzes the ring opening of oxepin-CoA and converts it to 3-oxo-5,6-dehydrosuberyl-CoA. Here we report the structures of PaaZ determined by electron cryomicroscopy with and without bound ligands. The structures reveal that three domain-swapped dimers of the enzyme form a trilobed structure. A combination of small-angle X-ray scattering (SAXS), computational studies, mutagenesis and microbial growth experiments suggests that the key intermediate is transferred from one active site to the other by a mechanism of electrostatic pivoting of the CoA moiety, mediated by a set of conserved positively charged residues.

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Active site dynamics in NADH oxidase from Thermus thermophilus studied by NMR spin relaxation

Journal of Biomolecular NMR ◽

10.1007/s10858-011-9542-0 ◽

2011 ◽

Vol 51 (1-2) ◽

pp. 71-82 ◽

Cited By ~ 2

Author(s):

Teresa Miletti ◽

Patrick J. Farber ◽

Anthony Mittermaier

Keyword(s):

Spin Relaxation ◽

Active Site ◽

Nadh Oxidase ◽

Thermus Thermophilus ◽

Nmr Spin Relaxation

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d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012936 ◽

2020 ◽

Vol 295 (23) ◽

pp. 7894-7904

Author(s):

Jordan L. Pederick ◽

Andrew P. Thompson ◽

Stephen G. Bell ◽

John B. Bruning

Keyword(s):

Binding Site ◽

Active Site ◽

Drug Target ◽

Catalytic Mechanism ◽

Thermus Thermophilus ◽

Monovalent Cation ◽

Antibiotic Drug ◽

Domains Of Life ◽

Alanine Dipeptide ◽

Insight Into

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine–d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl–d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+. Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

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