Structural Analysis of Actinorhodin Polyketide Ketoreductase: Cofactor Binding and Substrate Specificity

ABSTRACTThe organohalide-respiring bacteriumSulfurospirillum multivoransproduces a unique cobamide, namely, norpseudo-B12, which serves as cofactor of the tetrachloroethene (PCE) reductive dehalogenase (PceA). As previously reported, a replacement of the adeninyl moiety, the lower base of the cofactor, by exogenously applied 5,6-dimethylbenzimidazole led to inactive PceA. To explore the general effect of benzimidazoles on the PCE metabolism, the susceptibility of the organism for guided biosynthesis of various singly substituted benzimidazolyl-norcobamides was investigated, and their use as cofactor by PceA was analyzed. Exogenously applied 5-methylbenzimidazole (5-MeBza), 5-hydroxybenzimidazole (5-OHBza), and 5-methoxybenzimidazole (5-OMeBza) were found to be efficiently incorporated as lower bases into norcobamides (NCbas). Structural analysis of the NCbas by nuclear magnetic resonance spectroscopy uncovered a regioselectivity in the utilization of these precursors for NCba biosynthesis. When 5-MeBza was added, a mixture of 5-MeBza-norcobamide and 6-MeBza-norcobamide was formed, and the PceA enzyme activity was affected. In the presence of 5-OHBza, almost exclusively 6-OHBza-norcobamide was produced, while in the presence of 5-OMeBza, predominantly 5-OMeBza-norcobamide was detected. Both NCbas were incorporated into PceA, and no negative effect on the PceA activity was observed. In crystal structures of PceA, both NCbas were bound in the base-off mode with the 6-OHBza and 5-OMeBza lower bases accommodated by the same solvent-exposed hydrophilic pocket that harbors the adenine as the lower base of authentic norpseudo-B12. In this study, a selective production of different norcobamide isomers containing singly substituted benzimidazoles as lower bases is shown, and unique structural insights into their utilization as cofactors by a cobamide-containing enzyme are provided.IMPORTANCEGuided biosynthesis of norcobamides containing singly substituted benzimidazoles as lower bases by the organohalide-respiring epsilonproteobacteriumSulfurospirillum multivoransis reported. An unprecedented specificity in the formation of norcobamide isomers containing hydroxylated or methoxylated benzimidazoles was observed that implicated a strict regioselectivity of the norcobamide biosynthesis in the organism. In contrast to 5,6-dimethylbenzimidazolyl-norcobamide, the incorporation of singly substituted benzimidazolyl-norcobamides as a cofactor into the tetrachloroethene reductive dehalogenase was not impaired. The enzyme was found to be functional with different isomers and not limited to the use of adeninyl-norcobamide. Structural analysis of the enzyme equipped with either adeninyl- or benzimidazolyl-norcobamide cofactors visualized for the first time structurally different cobamides bound in base-off conformation to the cofactor-binding site of a cobamide-containing enzyme.

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A comparative structural analysis reveals distinctive features of co-factor binding and substrate specificity in plant aldo-keto reductases

Biochemical and Biophysical Research Communications ◽

10.1016/j.bbrc.2016.05.011 ◽

2016 ◽

Vol 474 (4) ◽

pp. 696-701 ◽

Cited By ~ 3

Author(s):

Priscila Oliveira de Giuseppe ◽

Marcelo Leite dos Santos ◽

Sylvia Morais de Sousa ◽

Karen E. Koch ◽

José Andrés Yunes ◽

...

Keyword(s):

Structural Analysis ◽

Substrate Specificity ◽

Distinctive Features ◽

Factor Binding

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Structures of trans-2-enoyl-CoA reductases from Clostridium acetobutylicum and Treponema denticola: insights into the substrate specificity and the catalytic mechanism

Biochemical Journal ◽

10.1042/bj20120871 ◽

2012 ◽

Vol 449 (1) ◽

pp. 79-89 ◽

Cited By ~ 6

Author(s):

Kuan Hu ◽

Meng Zhao ◽

Tianlong Zhang ◽

Manwu Zha ◽

Chen Zhong ◽

...

Keyword(s):

Substrate Specificity ◽

Clostridium Acetobutylicum ◽

Catalytic Mechanism ◽

Acyl Carrier Protein ◽

Substrate Binding ◽

Binding Domain ◽

Treponema Denticola ◽

Sequence Comparisons ◽

Cofactor Binding ◽

Catalytic Active Site

TERs (trans-2-enoyl-CoA reductases; EC 1.3.1.44), which specifically catalyse the reduction of crotonyl-CoA to butyryl-CoA using NADH as cofactor, have recently been applied in the design of robust synthetic pathways to produce butan-1-ol as a biofuel. We report in the present paper the characterization of a CaTER (a TER homologue in Clostridium acetobutylicum), the structures of CaTER in apo form and in complexes with NADH and NAD+, and the structure of TdTER (Treponema denticola TER) in complex with NAD+. Structural and sequence comparisons show that CaTER and TdTER share approximately 45% overall sequence identity and high structural similarities with the FabV class enoyl-acyl carrier protein reductases in the bacterial fatty acid synthesis pathway, suggesting that both types of enzymes belong to the same family. CaTER and TdTER function as monomers and consist of a cofactor-binding domain and a substrate-binding domain with the catalytic active site located at the interface of the two domains. Structural analyses of CaTER together with mutagenesis and biochemical data indicate that the conserved Glu75 determines the cofactor specificity, and the conserved Tyr225, Tyr235 and Lys244 play critical roles in catalysis. Upon cofactor binding, the substrate-binding loop changes from an open conformation to a closed conformation, narrowing a hydrophobic channel to the catalytic site. A modelling study shows that the hydrophobic channel is optimal in both width and length for the binding of crotonyl-CoA. These results provide molecular bases for the high substrate specificity and the catalytic mechanism of TERs.

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The Metalloid Reductase of Pseudomonas Moravenis Stanleyae Conveys Nanoparticle Mediated Metalloid Tolerance

10.26434/chemrxiv.6267383 ◽

2018 ◽

Author(s):

Richard Nemeth ◽

Mackenzie Neubert ◽

Thomas Ni ◽

Christopher J. Ackerson

Keyword(s):

Structural Analysis ◽

Substrate Specificity ◽

Glutathione Reductase ◽

Cell Lines ◽

Bacterial Cell ◽

Binding Pocket ◽

Oxidized Glutathione ◽

Se Nanoparticles ◽

In Cells

In the present work we have identified a glutathione reductase like metalloid reductase (GRLMR) responsible for mediating selenite tolerance in Pseudomonas moravenis stanleyae through the enzymatic generation of Se(0) nanoparticles. This enzyme has an unprecedented substrate specificity for selenodiglutathione (Km= 336 μM) over oxidized glutathione (Km=8.22 mM). This enzyme was able to induce selenite tolerance in foreign bacterial cell lines by increasing the IC90 for selenite from 1.9 mM in cell lacking the GRLMR gene to 21.3 mM for cells containing the GRLMR gene. It was later confirmed by STEM and EDS that Se nanoparticles were absent in control cells and present in cells expressing GRLMR. Structural analysis suggests the lack of a sulfur residue in the substrate/product binding pocket may be responsible for this unique substrate specificity.

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