The crystal structure of maleylacetate reductase fromRhizobiumsp. strain MTP-10005 provides insights into the reaction mechanism of enzymes in its original family

Abstract

Maleylacetate reductase plays a crucial role in catabolism of resorcinol by catalyzing the NAD(P)H-dependent reduction of maleylacetate, at a carbon-carbon double bond, to 3-oxoadipate. The crystal structure of maleylacetate reductase from Rhizobium sp. strain MTP-10005, GraC, has been elucidated by the X-ray diffraction method at 1.5 Å resolution. GraC is a homodimer, and each subunit consists of two domains: an N-terminal NADH-binding domain adopting an α/β structure and a C-terminal functional domain adopting an α-helical structure. Such structural features show similarity to those of the two existing families of enzymes in dehydroquinate synthase-like superfamily. However, GraC is distinct in dimer formation and activity expression mechanism from the families of enzymes. Two subunits in GraC have different structures from each other in the present crystal. One subunit has several ligands mimicking NADH and the substrate in the cleft and adopts a closed domain arrangement. In contrast, the other subunit does not contain any ligand causing structural changes and adopts an open domain arrangement. The structure of GraC reveals those of maleylacetate reductase both in the coenzyme, substrate-binding state and in the ligand-free state. The comparison of both subunit structures reveals a conformational change of the Tyr326 loop for interaction with His243 on ligand binding. Structures of related enzymes suggest that His243 is likely a catalytic residue of GraC. Mutational analyses of His243 and Tyr326 support the catalytic roles proposed from structural information. The crystal structure of GraC characterizes the maleylacetate reductase family as a third family in the dehydroquinate synthase-like superfamily. Proteins 2016; 84:1029-1042. © 2016 Wiley Periodicals, Inc.

Residues His172 and Lys238 are Essential for the Catalytic Activity of the Maleylacetate Reductase from Sphingobium chlorophenolicum Strain L-1

“…The reduced catalytic efficiency for K140A, H237A, H241A and H251A mutants is likely due to decreased substrate binding resulted from the loss of ionic interactions between the positively charged side chains of these basic residues and the carboxylate groups of MA. Our results are also consistent with the enzymatic study on Rm -MR with the H243A mutant (corresponding to H241A mutant of PcpE), which exhibited reduced catalytic activity 30 . Further studies are warranted to identify whether mutation of these residues to other basic amino acids, such K140R, H237R, H251K and H251R, could improve the catalytic efficiency of PcpE.…”

“…However, it is difficult to validate the accuracy of our docking result since the MA binding site has never been experimentally identified for any maleylacetate reductase. The putative 2-CMA/MA binding site for PcpE from our current docking study is located at the same site proposed for Rm -MR based on the binding site of a sulfate anion 30 , implying that PcpE model is likely to be reasonable to guide our subsequent mutagenesis and kinetics studies. Although other amino acid residues may also be involved in 2-CMA/MA binding, we were more interested in identifying whether any of the seven basic amino acid residues plays an essential role in the reductive reactions catalyzed by PcpE.…”

“…Recently, the crystal structure of the maleylacetate reductase from Rhizobium sp. strain MTP-10005 ( Rm -MR, PDB ID code: 3W5S), which shares 52% sequence identity with PcpE, was determined 30 . Retrospectively, we performed a comparison study of the PcpE model and the Rm -MR structure to further validate the quality of our homology/comparative modeling.…”

Diversity and Evolutionary Analysis of Iron-Containing (Type-III) Alcohol Dehydrogenases in Eukaryotes

“…Interestingly, maleylacetate reductases (MAR subfamily; cd08177) are active in absence of metal ions, and do not have a divalent metal M 2+ at their active center [69,95–97]; this may be due to the substitution of Asp242 by asparagine or arginine (Figs 5–7), which probably makes that MAR enzymes lose affinity for metal ions [95]. Considering this, members of the uncharacterized subfamily FeADH2 (cd08183), and some members of the HEPD subfamily (cd08182), probably are also functional in absence of divalent metal because Asp242 is replaced by glutamine in these proteins (Fig 7).…”

“…coli FucO showed that His267 is not coordinated with Fe 2+ ions [59]. Recently, Fujii et al, [95] performed an structural comparison between E . coli FucO and Rhizobium sp.…”