The catalytic mechanism of Pseudomonas aeruginosa cd1 nitrite reductase

2011 ◽  
Vol 39 (1) ◽  
pp. 195-200 ◽  
Author(s):  
Serena Rinaldo ◽  
Giorgio Giardina ◽  
Nicoletta Castiglione ◽  
Valentina Stelitano ◽  
Francesca Cutruzzolà
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Product Inhibition ◽  
Electron Donors ◽  
Nitrite Reduction ◽  
Nitrite Reductases ◽  
Shed Light ◽  
Denitrification Process

The cd1 NiRs (nitrite reductases) are enzymes catalysing the reduction of nitrite to NO (nitric oxide) in the bacterial energy conversion denitrification process. These enzymes contain two distinct redox centres: one covalently bound c-haem, which is reduced by external electron donors, and another peculiar porphyrin, the d1-haem (3,8-dioxo-17-acrylate-porphyrindione), where nitrite is reduced to NO. In the present paper, we summarize the most recent results on the mechanism of nitrite reduction by the cd1 NiR from Pseudomonas aeruginosa. We discuss the essential catalytic features of this enzyme, with special attention to the allosteric regulation of the enzyme's activity and to the mechanism employed to avoid product inhibition, i.e. trapping of the active-site reduced haem by the product NO. These results shed light on the reactivity of cd1 NiRs and assign a central role to the unique d1-haem, present only in this class of enzymes.

New insights into the activity of Pseudomonas aeruginosa cd1 nitrite reductase

2008 ◽  
Vol 36 (6) ◽  
pp. 1155-1159 ◽  
Author(s):  
Serena Rinaldo ◽  
Alessandro Arcovito ◽  
Giorgio Giardina ◽  
Nicoletta Castiglione ◽  
Maurizio Brunori ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Nitrite Reductase ◽  
Nitrite Reduction ◽  
Nitrite Reductases ◽  
Cytochrome Cd1 ◽  
Haem Iron ◽  
Shed Light ◽  
Denitrification Process

The cytochrome cd1 nitrite reductases are enzymes that catalyse the reduction of nitrite to nitric oxide (NO) in the bacterial energy conversion denitrification process. These enzymes contain two different redox centres: one covalently bound c-haem, which is reduced by external donors, and one peculiar d1-haem, where catalysis occurs. In the present paper, we summarize the current understanding of the reaction of nitrite reduction in the light of the most recent results on the enzyme from Pseudomonas aeruginosa and discuss the differences between enzymes from different organisms. We have evidence that release of NO from the ferrous d1-haem occurs rapidly enough to be fully compatible with the turnover, in contrast with previous hypotheses, and that the substrate nitrite is able to displace NO from the d1-haem iron. These results shed light on the mechanistic details of the activity of cd1 nitrite reductases and on the biological role of the d1-haem, whose presence in this class of enzymes has to date been unexplained.

scholarly journals Structure of heme d 1-free cd 1 nitrite reductase NirS

2020 ◽  
Vol 76 (6) ◽  
pp. 250-256
Author(s):  
Thomas Klünemann ◽  
Wulf Blankenfeldt
Keyword(s):  
Nitric Oxide ◽  
Pseudomonas Aeruginosa ◽  
Nitrite Reductase ◽  
Active Site ◽  
Opportunistic Pathogen ◽  
Active Enzyme ◽  
Free Form ◽  
Nitrate Respiration ◽  
Gram Negative ◽  
Open Conformation

A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by the cd 1 nitrite reductase NirS in, for example, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d 1 as an essential part of its active site. Although NirS has been well studied mechanistically and structurally, the focus of previous studies has been on the active heme d 1-bound form. The heme d 1-free form of NirS reported here, which represents a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, the movement of a loop around Trp498 seems to be related to a widening of the propeller, allowing easier access to the heme d 1-binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed.

scholarly journals Structural studies reveal flexible roof of active site responsible for ω-transaminase CrmG overcoming by-product inhibition

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Jinxin Xu ◽  
Xiaowen Tang ◽  
Yiguang Zhu ◽  
Zhijun Yu ◽  
Kai Su ◽  
...  
Keyword(s):  
Pharmaceutical Industry ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Product Inhibition ◽  
Strong Interactions ◽  
Structural Studies ◽  
Biochemical Studies ◽  
Fundamental Challenge ◽  
Amine Compounds

AbstractAmine compounds biosynthesis using ω-transaminases has received considerable attention in the pharmaceutical industry. However, the application of ω-transaminases was hampered by the fundamental challenge of severe by-product inhibition. Here, we report that ω-transaminase CrmG from Actinoalloteichus cyanogriseus WH1-2216-6 is insensitive to inhibition from by-product α-ketoglutarate or pyruvate. Combined with structural and QM/MM studies, we establish the detailed catalytic mechanism for CrmG. Our structural and biochemical studies reveal that the roof of the active site in PMP-bound CrmG is flexible, which will facilitate the PMP or by-product to dissociate from PMP-bound CrmG. Our results also show that amino acceptor caerulomycin M (CRM M), but not α-ketoglutarate or pyruvate, can form strong interactions with the roof of the active site in PMP-bound CrmG. Based on our results, we propose that the flexible roof of the active site in PMP-bound CrmG may facilitate CrmG to overcome inhibition from the by-product.

scholarly journals Structural basis for glucose tolerance in GH1 β-glucosidases

2014 ◽  
Vol 70 (6) ◽  
pp. 1631-1639 ◽  
Author(s):  
Priscila Oliveira de Giuseppe ◽  
Tatiana de Arruda Campos Brasil Souza ◽  
Flavio Henrique Moreira Souza ◽  
Leticia Maria Zanphorlin ◽  
Carla Botelho Machado ◽  
...  
Keyword(s):  
Glucose Tolerance ◽  
Active Site ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Product Inhibition ◽  
Structural Basis ◽  
Clear Correlation ◽  
Biomass Saccharification ◽  
Glucose Tolerant ◽  
Shed Light

Product inhibition of β-glucosidases (BGs) by glucose is considered to be a limiting step in enzymatic technologies for plant-biomass saccharification. Remarkably, some β-glucosidases belonging to the GH1 family exhibit unusual properties, being tolerant to, or even stimulated by, high glucose concentrations. However, the structural basis for the glucose tolerance and stimulation of BGs is still elusive. To address this issue, the first crystal structure of a fungal β-glucosidase stimulated by glucose was solved in native and glucose-complexed forms, revealing that the shape and electrostatic properties of the entrance to the active site, including the +2 subsite, determine glucose tolerance. The aromatic Trp168 and the aliphatic Leu173 are conserved in glucose-tolerant GH1 enzymes and contribute to relieving enzyme inhibition by imposing constraints at the +2 subsite that limit the access of glucose to the −1 subsite. The GH1 family β-glucosidases are tenfold to 1000-fold more glucose tolerant than GH3 BGs, and comparative structural analysis shows a clear correlation between active-site accessibility and glucose tolerance. The active site of GH1 BGs is located in a deep and narrow cavity, which is in contrast to the shallow pocket in the GH3 family BGs. These findings shed light on the molecular basis for glucose tolerance and indicate that GH1 BGs are more suitable than GH3 BGs for biotechnological applications involving plant cell-wall saccharification.

scholarly journals Spectroscopic and computational studies of reversible O2 binding by a cobalt complex of relevance to cysteine dioxygenase

2017 ◽  
Vol 46 (39) ◽  
pp. 13229-13241 ◽  
Author(s):  
Anne A. Fischer ◽  
Sergey V. Lindeman ◽  
Adam T. Fiedler
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Cobalt Complex ◽  
Computational Studies ◽  
Cysteine Dioxygenase ◽  
Shed Light ◽  
O2 Binding

Spectroscopic and computational studies of reversible O2 binding by a cobalt active-site mimic shed light on the catalytic mechanism of cysteine dioxygenases.

scholarly journals Structures of the hydrolase domain of zebrafish 10-formyltetrahydrofolate dehydrogenase and its complexes reveal a complete set of key residues for hydrolysis and product inhibition

2015 ◽  
Vol 71 (4) ◽  
pp. 1006-1021 ◽  
Author(s):  
Chien-Chih Lin ◽  
Phimonphan Chuankhayan ◽  
Wen-Ni Chang ◽  
Tseng-Ting Kao ◽  
Hong-Hsiang Guan ◽  
...  
Keyword(s):  
Binding Site ◽  
Active Site ◽  
Catalytic Mechanism ◽  
Product Inhibition ◽  
Product Specificity ◽  
Product Release ◽  
Terminal Domain ◽  
Complete Set ◽  
Formyltetrahydrofolate Dehydrogenase ◽  
Key Residues

10-Formyltetrahydrofolate dehydrogenase (FDH), which is composed of a small N-terminal domain (Nt-FDH) and a large C-terminal domain, is an abundant folate enzyme in the liver and converts 10-formyltetrahydrofolate (10-FTHF) to tetrahydrofolate (THF) and CO2. Nt-FDH alone possesses a hydrolase activity, which converts 10-FTHF to THF and formate in the presence of β-mercaptoethanol. To elucidate the catalytic mechanism of Nt-FDH, crystal structures of apo-form zNt-FDH from zebrafish and its complexes with the substrate analogue 10-formyl-5,8-dideazafolate (10-FDDF) and with the products THF and formate have been determined. The structures reveal that the conformations of three loops (residues 86–90, 135–143 and 200–203) are altered upon ligand (10-FDDF or THF) binding in the active site. The orientations and geometries of key residues, including Phe89, His106, Arg114, Asp142 and Tyr200, are adjusted for substrate binding and product release during catalysis. Among them, Tyr200 is especially crucial for product release. An additional potential THF binding site is identified in the cavity between two zNt-FDH molecules, which might contribute to the properties of product inhibition and THF storage reported for FDH. Together with mutagenesis studies and activity assays, the structures of zNt-FDH and its complexes provide a coherent picture of the active site and a potential THF binding site of zNt-FDH along with the substrate and product specificity, lending new insights into the molecular mechanism underlying the enzymatic properties of Nt-FDH.

scholarly journals A QM/MM Study of Nitrite Binding Modes in a Three-Domain Heme-Cu Nitrite Reductase

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2997
Author(s):  
Kakali Sen ◽  
Michael Hough ◽  
Richard Strange ◽  
Chin Yong ◽  
Thomas Keal
Keyword(s):  
Nitric Oxide ◽  
Nitrite Reductase ◽  
Binding Mode ◽  
Nitrite Reduction ◽  
Quantum Mechanical ◽  
Binding Modes ◽  
Crystallographic Study ◽  
Nitrite Reductases ◽  
Global Nitrogen Cycle ◽  
Oxygen Mode

Copper-containing nitrite reductases (CuNiRs) play a key role in the global nitrogen cycle by reducing nitrite (NO2−) to nitric oxide, a reaction that involves one electron and two protons. In typical two-domain CuNiRs, the electron is acquired from an external electron-donating partner. The recently characterised Rastonia picketti (RpNiR) system is a three-domain CuNiR, where the cupredoxin domain is tethered to a heme c domain that can function as the electron donor. The nitrite reduction starts with the binding of NO2− to the T2Cu centre, but very little is known about how NO2− binds to native RpNiR. A recent crystallographic study of an RpNiR mutant suggests that NO2− may bind via nitrogen rather than through the bidentate oxygen mode typically observed in two-domain CuNiRs. In this work we have used combined quantum mechanical/molecular mechanical (QM/MM) methods to model the binding mode of NO2− with native RpNiR in order to determine whether the N-bound or O-bound orientation is preferred. Our results indicate that binding via nitrogen or oxygen is possible for the oxidised Cu(II) state of the T2Cu centre, but in the reduced Cu(I) state the N-binding mode is energetically preferred.

scholarly journals An unprecedented insight into the catalytic mechanism of copper nitrite reductase from atomic-resolution and damage-free structures

2021 ◽  
Vol 7 (1) ◽  
pp. eabd8523
Author(s):  
Samuel L. Rose ◽  
Svetlana V. Antonyuk ◽  
Daisuke Sasaki ◽  
Keitaro Yamashita ◽  
Kunio Hirata ◽  
...  
Keyword(s):  
Bound States ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Atomic Resolution ◽  
Reaction Intermediates ◽  
X Ray ◽  
Nitrite Reductases ◽  
Radiation Induced ◽  
Insight Into ◽  
Shed Light

Copper-containing nitrite reductases (CuNiRs), encoded by nirK gene, are found in all kingdoms of life with only 5% of CuNiR denitrifiers having two or more copies of nirK. Recently, we have identified two copies of nirK genes in several α-proteobacteria of the order Rhizobiales including Bradyrhizobium sp. ORS 375, encoding a four-domain heme-CuNiR and the usual two-domain CuNiR (Br2DNiR). Compared with two of the best-studied two-domain CuNiRs represented by the blue (AxNiR) and green (AcNiR) subclasses, Br2DNiR, a blue CuNiR, shows a substantially lower catalytic efficiency despite a sequence identity of ~70%. Advanced synchrotron radiation and x-ray free-electron laser are used to obtain the most accurate (atomic resolution with unrestrained SHELX refinement) and damage-free (free from radiation-induced chemistry) structures, in as-isolated, substrate-bound, and product-bound states. This combination has shed light on the protonation states of essential catalytic residues, additional reaction intermediates, and how catalytic efficiency is modulated.

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