Crystal Structure and Comparative Functional Analyses of a Mycobacterium Aldo-Keto Reductase

Scoble, Judith A.; McAlister, Adrian Dale; Fulton, Zara; Troy, S.; Byres, E.; Vivian, J.P.; Brammananth, Rajini; Wilce, Matthew Charles James; Nours, Jérôme Le; Zaker-Tabrizi, Leyla; Coppel, Ross L.; Crellin, Paul K.; Rossjohn, Jamie; Beddoe, Travis

doi:10.1016/j.jmb.2010.02.021

Cited by 11 publications

(13 citation statements)

References 49 publications

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“…The gene encoding Rv2971 was cloned, overexpressed and purified as soluble recombinant protein as previously described (Scoble et al, 2010). For crystallization experiments, the purified recombinant protein was buffer-exchanged into 10 mM Tris-HCl pH 8.0, 200 mM NaCl and concentrated to 7 mg ml À1 .…”

Section: Cloning Expression and Purification Of Recombinant Proteinmentioning

confidence: 99%

“…The coordinates of M. smegmatis AKR5H1 MSMEG_2407 (67% sequence identity; PDB entry 2wzm; Scoble et al, 2010) were used as the search model. The resultant LLG score and TZF score obtained were 2010.15 and 43.8, respectively.…”

Section: Structural Determinationmentioning

confidence: 99%

“…To gain functional insight into the role of AKR5H1, the crystal structure of MSMEG_2407 has been determined in its apo form and in its holoenzyme form in the presence of NADPH (Scoble et al, 2010). The crystal structure reveals an (/) 8 -barrel topology, or a TIM-barrel fold, typical of other AKRs as previously described (Banner et al, 1975), and enabled residues essential for enzyme kinetic activity and cofactor binding to be ascertained.…”

Section: Introductionmentioning

confidence: 98%

“…rv2971 has previously been characterized as an essential gene for growth and survival in M. tuberculosis (Sassetti et al, 2003) with a potential role in the detoxification of toxic metabolites (Grimshaw, 1992;Grant et al, 2003;Penning & Drury, 2007). Recently, the catalytic activity of Rv2971 and its M. smegmatis orthologue MSMEG_2407 (67% sequence identity) has been characterized, revealing differences in dicarbonyl substrate specificities and affinities (Scoble et al, 2010). The potency of INH was characterized against both enzymes, revealing a higher affinity against MSMEG_2407 compared with Rv2971, with apparent K i values of 6.0 AE 1.2 and 31.0 AE 1.4 mM reported, respectively (Scoble et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

“…Recently, the catalytic activity of Rv2971 and its M. smegmatis orthologue MSMEG_2407 (67% sequence identity) has been characterized, revealing differences in dicarbonyl substrate specificities and affinities (Scoble et al, 2010). The potency of INH was characterized against both enzymes, revealing a higher affinity against MSMEG_2407 compared with Rv2971, with apparent K i values of 6.0 AE 1.2 and 31.0 AE 1.4 mM reported, respectively (Scoble et al, 2010).…”

Section: Introductionmentioning

confidence: 99%

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A structural characterization of the isoniazidMycobacterium tuberculosisdrug target, Rv2971, in its unliganded form

et al. 2014

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Aldo-keto reductases (AKR) are a large superfamily of NADPH-dependent oxidoreductases and play a role in detoxification of toxic metabolites. Rv2971, an AKR inMycobacterium tuberculosis, has recently been identified as a target of isoniazid, a key first-line drug against tuberculosis. Here, the cloning, expression, purification, crystallization and structural characterization of Rv2971 are described. To gain insight into its function, the crystal structure of Rv2971 was successfully determined to 1.60 Å resolution in its unliganded form. The structure exhibits a TIM-barrel fold typical of AKRs, revealing structural characteristics essential for function and substrate specificities, allowing a structural comparison between Rv2971 and other mycobacterial AKRs.

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Section: Cloning Expression and Purification Of Recombinant Proteinmentioning

confidence: 99%

Section: Structural Determinationmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

A structural characterization of the isoniazidMycobacterium tuberculosisdrug target, Rv2971, in its unliganded form

et al. 2014

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Structural and mutational studies on an aldo‐keto reductase AKR5C3 from Gluconobacter oxydans

et al. 2014

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An aldo-keto reductase AKR5C3 from Gluconobacter oxydans (designated as Gox0644) is a useful enzyme with various substrates, including aldehydes, diacetyl, keto esters, and aketocarbonyl compounds. The crystal structures of AKR5C3 in apoform in complex with NADPH and the D53A mutant (AKR5C3 -D53A ) in complex with NADPH are presented herein. Structure comparison and site-directed mutagenesis combined with biochemical kinetics analysis reveal that the conserved Asp53 in the AKR5C3 catalytic tetrad has a crucial role in securing active pocket conformation. The gain-of-function Asp53 to Ala mutation triggers conformational changes on the Trp30 and Trp191 side chains, improving NADPH affinity to AKR5C3, which helps increase catalytic efficiency. The highly conserved Trp30 and Trp191 residues interact with the nicotinamide moiety of NADPH and help form the NADPH-binding pocket. The AKR5C3 -W30A and AKR5C3 -W191Y mutants show decreased activities, confirming that both residues facilitate catalysis. Residue Trp191 is in the loop structure, and the AKR5C3 -W191Y mutant does not react with benzaldehyde, which might also determine substrate recognition. Arg192, which is involved in the substrate binding, is another important residue. The introduction of R192G increases substrate-binding affinity by improving hydrophobicity in the substrate-binding pocket. These results not only supplement the AKRs superfamily with crystal structures but also provide useful information for understanding the catalytic properties of AKR5C3 and guiding further engineering of this enzyme.

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Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding

Qin

Yamamura

Miyakawa

et al. 2013

Appl Microbiol Biotechnol

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Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

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Crystal Structure and Comparative Functional Analyses of a Mycobacterium Aldo-Keto Reductase

Cited by 11 publications

References 49 publications

A structural characterization of the isoniazidMycobacterium tuberculosisdrug target, Rv2971, in its unliganded form

A structural characterization of the isoniazidMycobacterium tuberculosisdrug target, Rv2971, in its unliganded form

Structural and mutational studies on an aldo‐keto reductase AKR5C3 from Gluconobacter oxydans

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding

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