Unique coupling of mono- and dioxygenase chemistries in a single active site promotes heme degradation

Bacterial pathogens must acquire host iron for survival and colonization. Because free iron is restricted in the host, numerous pathogens have evolved to overcome this limitation by using a family of monooxygenases that mediate the oxidative cleavage of heme into biliverdin, carbon monoxide, and iron. However, the etiological agent of tuberculosis, Mycobacterium tuberculosis, accomplishes this task without generating carbon monoxide, which potentially induces its latent state. Here we show that this unusual heme degradation reaction proceeds through sequential mono- and dioxygenation events within the single active center of MhuD, a mechanism unparalleled in enzyme catalysis. A key intermediate of the MhuD reaction is found to be meso-hydroxyheme, which reacts with O2 at an unusual position to completely suppress its monooxygenation but to allow ring cleavage through dioxygenation. This mechanistic change, possibly due to heavy steric deformation of hydroxyheme, rationally explains the unique heme catabolites of MhuD. Coexistence of mechanistically distinct functions is a previously unidentified strategy to expand the physiological outcome of enzymes, and may be applied to engineer unique biocatalysts.

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Intermolecular C–H Amidation of Alkenes with Carbon Monoxide and Azides via Tandem Palladium Catalysis

Synthesis ◽

10.1055/a-1401-4486 ◽

2021 ◽

Author(s):

Zheng-Yang Gu ◽

Yang Wu ◽

Feng Jin ◽

Bao Xiaoguang ◽

Ji-Bao Xia

Keyword(s):

Carbon Monoxide ◽

Palladium Catalysis ◽

Computational Studies ◽

Organic Azides ◽

Reaction Proceeds ◽

Palladium Catalyzed ◽

Directing Group ◽

And Control

An atom- and step-economic intermolecular multi-component palladium-catalyzed C–H amidation of alkenes with carbon monoxide and organic azides has been developed for the synthesis of alkenyl amides. The reaction proceeds efficiently without an ortho-directing group on the alkene substrates. Nontoxic dinitrogen is generated as the sole by-product. Computational studies and control experiments have revealed that the reaction takes place via an unexpected mechanism by tandem palladium catalysis.

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Studies on active site mutants of P. falciparum adenylosuccinate synthetase: Insights into enzyme catalysis and activation

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics ◽

10.1016/j.bbapap.2010.07.015 ◽

2010 ◽

Vol 1804 (10) ◽

pp. 1996-2002 ◽

Cited By ~ 4

Author(s):

Sonali Mehrotra ◽

B.N. Mylarappa ◽

Prathima Iyengar ◽

Hemalatha Balaram

Keyword(s):

Active Site ◽

Enzyme Catalysis ◽

Adenylosuccinate Synthetase ◽

Active Site Mutants

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Reaction of Carbon Monoxide with the Reduced Active Site of Bacterial Nitric Oxide Reductase†

Biochemistry ◽

10.1021/bi011428t ◽

2001 ◽

Vol 40 (44) ◽

pp. 13361-13369 ◽

Cited By ~ 27

Author(s):

Janneke H. M. Hendriks ◽

Louise Prior ◽

Adam R. Baker ◽

Andrew J. Thomson ◽

Matti Saraste ◽

...

Keyword(s):

Nitric Oxide ◽

Carbon Monoxide ◽

Active Site ◽

Nitric Oxide Reductase

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The thermal degradation of μ-Diphenylacetylene-bis(tricarbonylcobalt)

Australian Journal of Chemistry ◽

10.1071/ch9731791 ◽

1973 ◽

Vol 26 (8) ◽

pp. 1791 ◽

Cited By ~ 2

Author(s):

RS Dickson ◽

LJ Michel

Keyword(s):

Differential Scanning Calorimetry ◽

Thermal Decomposition ◽

Carbon Monoxide ◽

Thermal Degradation ◽

Organic Compounds ◽

Degradation Mechanism ◽

Scanning Calorimetry ◽

Organic Products ◽

Suitable Temperature ◽

Degradation Reaction

The thermal decomposition of Co2(CO)6(PhC2Ph) has been investigated in detail. Differential scanning calorimetry was used to determine the most suitable temperature range for the study. At 180�, Co2(CO)6(PhC2Ph) decomposes to form cobalt, carbon monoxide, tetraphenylcyclopentadienone, hexaphenylbenzene, and other organic compounds. Variation in the temperature, the time, and the solvent used for the degradation reaction causes significant changes in the yields of the organic products. An investigation of the effects of adding stoichiometric amounts of free alkyne, tetra-phenylcyclopentadienone, and hexaphenylbenzene has been initiated in an attempt to understand the degradation mechanism.

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Coordination structure of active site in synthetic hemoprotein (albumin-heme) with dioxygen and carbon monoxide

Macromolecular Symposia ◽

10.1002/masy.200390134 ◽

2003 ◽

Vol 195 (1) ◽

pp. 275-280

Author(s):

Eishun Tsuchida ◽

Akito Nakagawa ◽

Teruyuki Komatsu

Keyword(s):

Carbon Monoxide ◽

Active Site ◽

Coordination Structure

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Crystal structure of hexanoyl-CoA bound to β-ketoacyl reductase FabG4 of Mycobacterium tuberculosis

Biochemical Journal ◽

10.1042/bj20121107 ◽

2013 ◽

Vol 450 (1) ◽

pp. 127-139 ◽

Cited By ~ 27

Author(s):

Debajyoti Dutta ◽

Sudipta Bhattacharyya ◽

Amlan Roychowdhury ◽

Rupam Biswas ◽

Amit Kumar Das

Keyword(s):

Mycobacterium Tuberculosis ◽

Active Site ◽

Ternary Complex ◽

Catalytic Mechanism ◽

Acyl Carrier Protein ◽

Structural Data ◽

Acid Synthesis ◽

Binary Complex ◽

C Terminus ◽

Covalently Linked

FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into β-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal ‘flavodoxintype’ domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4–NADH binary complex and the FabG4–NAD+–hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

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Structural insight into conformational dynamics of non-active site mutations in KasA: A Mycobacterium tuberculosis target protein

Gene ◽

10.1016/j.gene.2019.144082 ◽

2019 ◽

Vol 720 ◽

pp. 144082 ◽

Cited By ~ 2

Author(s):

Manikandan Jayaraman ◽

Savita Kumari Rajendra ◽

Krishna Ramadas

Keyword(s):

Mycobacterium Tuberculosis ◽

Active Site ◽

Conformational Dynamics ◽

Target Protein ◽

Structural Insight ◽

Insight Into ◽

Active Site Mutations

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Structure, interactions and action of Mycobacterium tuberculosis 3-hydroxyisobutyric acid dehydrogenase

Biochemical Journal ◽

10.1042/bcj20180271 ◽

2018 ◽

Vol 475 (15) ◽

pp. 2457-2471 ◽

Cited By ~ 1

Author(s):

Rajapiramuthu Srikalaivani ◽

Amrita Singh ◽

Mamannamana Vijayan ◽

Avadhesha Surolia

Keyword(s):

Mycobacterium Tuberculosis ◽

Active Site ◽

Mutational Analysis ◽

Gel Filtration ◽

Ph Optimum ◽

Native Page ◽

Acid Dehydrogenase ◽

Binary Complexes ◽

Solution Studies ◽

Hydroxyisobutyric Acid

Biochemical and crystallographic studies on Mycobacterium tuberculosis 3-hydroxyisobutyric acid dehydrogenase (MtHIBADH), a member of the 3-hydroxyacid dehydrogenase superfamily, have been carried out. Gel filtration and blue native PAGE of MtHIBADH show that the enzyme is a dimer. The enzyme preferentially uses NAD+ as the cofactor and is specific to S-hydroxyisobutyric acid (HIBA). It can also use R-HIBA, l-serine and 3-hydroxypropanoic acid (3-HP) as substrates, but with much less efficiency. The pH optimum for activity is ∼11. Structures of the native enzyme, the holoenzyme, binary complexes with NAD+, S-HIBA, R-HIBA, l-serine and 3-HP and ternary complexes involving the substrates and NAD+ have been determined. None of the already known structures of HIBADH contain a substrate molecule at the binding site. The structures reported here provide for the first time, among other things, a clear indication of the location and interactions of the substrates at the active site. They also define the entrance of the substrates to the active site region. The structures provide information on the role of specific residues at the active site and the entrance. The results obtained from crystal structures are consistent with solution studies including mutational analysis. They lead to the proposal of a plausible mechanism of the action of the enzyme.

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