Database accession: MF7000445
Name: NAD-binding-2 domain-containing protein (mutant)
PDB ID: 8a5z
Experimental method: X-ray (2.31 Å)
Assembly: Homodimer
Source organism: Ensifer adhaerens
Primary publication of the structure:
Gilio AK, Thorpe TW, Heyam A, Petchey MR, Pogrányi B, France SP, Howard RM, Karmilowicz MJ, Lewis R, Turner N, Grogan G
A Reductive Aminase Switches to Imine Reductase Mode for a Bulky Amine Substrate.
(2023) ACS Catalysis 13: 1669-1677
PMID: 36776386
Abstract:
Imine reductases (IREDs) catalyze the asymmetric reduction of cyclic imines, but also in some cases the coupling of ketones and amines to form secondary amine products in an enzyme-catalyzed reductive amination (RedAm) reaction. Enzymatic RedAm reactions have typically used small hydrophobic amines, but many interesting pharmaceutical targets require that larger amines be used in these coupling reactions. Following the identification of IR77 from Ensifer adhaerens as a promising biocatalyst for the reductive amination of cyclohexanone with pyrrolidine, we have characterized the ability of this enzyme to catalyze couplings with larger bicyclic amines such as isoindoline and octahydrocyclopenta(c)pyrrole. By comparing the activity of IR77 with reductions using sodium cyanoborohydride in water, it was shown that, while the coupling of cyclohexanone and pyrrolidine involved at least some element of reductive amination, the amination with the larger amines likely occurred ex situ, with the imine recruited from solution for enzyme reduction. The structure of IR77 was determined, and using this as a basis, structure-guided mutagenesis, coupled with point mutations selecting improving amino acid sites suggested by other groups, permitted the identification of a mutant A208N with improved activity for amine product formation. Improvements in conversion were attributed to greater enzyme stability as revealed by X-ray crystallography and nano differential scanning fluorimetry. The mutant IR77-A208N was applied to the preparative scale amination of cyclohexanone at 50 mM concentration, with 1.2 equiv of three larger amines, in isolated yields of up to 93%.
Annotations from the GeneOntology database. Only terms that fit at least two of the interacting proteins are shown. Molecular function:
3-hydroxybutyrate dehydrogenase activity 3-hydroxybutyrate dehydrogenase activity
methylated histone binding methylated histone binding
NADP binding NADP binding
Biological process:
cellular catabolic process cellular catabolic process
Cellular component:
cytosol cytosol
Structural annotations of the participating protein chains.Entry contents: 2 distinct polypeptide molecules
Chains: A, B
Notes: All chains according to the most probable oligomerization state stored in PDBe were considered.
Number of unique protein segments: 1
Name: Uncharacterized protein
Source organism: Ensifer adhaerens
Length: 296 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMKPSISVLGTGRMGSALARALLQAGYRTVVWNRTSEKAEPLAALGATVAPTVRQAIDASGIVIVNVSDYAATSTLLRASDVTPGLRGKLIVELTSGTPEGARETSQWTAAHGARYLDGAILATPDFIGTDAGTILLSGALEPFAANEDVFRALGGNVQHIGTEPGLANALDSAVLALMWGALFGGLHAIAVCRAEEIDLGELGRQWAATAPVVEGLVADLIKRTSAGRFVSDAETLSSISPHYGAFQHLKELMEARRIDRTVVDGYDAIFRRAIASGHLHDDFAALSQFMGKAEQP
UniProtKB AC: A0A0L8BGL4 (positions: 2-293)
Coverage: 98%
Name: Uncharacterized protein
Source organism: Ensifer adhaerens
Length: 296 residues
Sequence:Sequence according to the corresponding UniProt protein segmentMKPSISVLGTGRMGSALARALLQAGYRTVVWNRTSEKAEPLAALGATVAPTVRQAIDASGIVIVNVSDYAATSTLLRASDVTPGLRGKLIVELTSGTPEGARETSQWTAAHGARYLDGAILATPDFIGTDAGTILLSGALEPFAANEDVFRALGGNVQHIGTEPGLANALDSAVLALMWGALFGGLHAIAVCRAEEIDLGELGRQWAATAPVVEGLVADLIKRTSAGRFVSDAETLSSISPHYGAFQHLKELMEARRIDRTVVDGYDAIFRRAIASGHLHDDFAALSQFMGKAEQP
UniProtKB AC: A0A0L8BGL4 (positions: 1-295)
Coverage: 99%
Evidence demonstrating that the participating proteins are unstructured prior to the interaction and their folding is coupled to binding. Representative domain in related structures: -
Evidence level: Insufficient evidence (candidate)
Evidence coverage: Only some parts of the structure participates in mutual synergistic folding.
Complex Evidence:
There is no information about the monomeric forms of the constituting partners. The C-terminal part is an intertwined, all-helical dimerization domain, while the N-terminal NADP-binding domain is well-folded.
Chain A:
N/A
Chain B:
N/A
Surface and contacts features:
Structures from the PDB that contain the same number of proteins, and the proteins from the two structures show a sufficient degree of pairwise similarity, i.e. they belong to the same UniRef90 cluster (the full proteins exhibit at least 90% sequence identity) and convey roughly the same region to their respective interactions (the two regions from the two proteins share a minimum of 70% overlap). No related structure was found in the MFIB database.
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