The cyanobacterial aldehyde deformylating oxygenase (cADO) is a key enzyme that catalyzes the unusual deformylation of aliphatic aldehydes for alkane biosynthesis and can be applied to the production of biofuel in vitro and in vivo. In this study, we determined crystal structures of two ADOs from Limnothrix sp. KNUA012 (LiADO) and Oscillatoria sp. KNUA011 (OsADO). The structures of LiADO and OsADO resembled those of typical cADOs, consisting of eight α-helices found in ferritin-like di-iron proteins. However, structural comparisons revealed that while the LiADO active site was vacant of iron and substrates, the OsADO active site was fully occupied, containing both a coordinated metal ion and substrate. Previous reports indicated that helix 5 is capable of adopting two distinct conformations depending upon the existence of bound iron. We observed that helix 5 of OsADO with an iron bound in the active site presented as a long helix, whereas helix 5 of LiADO, which lacked iron in the active site, presented two conformations (one long and two short helices), indicating that an equilibrium exists between the two states in solution. Furthermore, acquisition of a structure having a fully occupied active site is unique in the absence of higher iron concentrations as compared with other cADO structures, wherein low affinity for iron complicates the acquisition of crystal structures with bound iron. An in-depth analysis of the ADO apo-enzyme, the enzyme with substrate bound, and the enzyme with both iron and substrate bound provided novel insight into substrate-binding modes in the absence of a coordinated metal ion and suggested a separate two-step binding mechanism for substrate and iron co-factors. Moreover, our results provided a comprehensive structural basis for conformational changes induced by binding of the substrate and co-factor.

Fumarylacetoacetate hydrolase (FAH) is essential for the degradation of aromatic amino acids as well as for the cleavage of carbon-carbon bonds in metabolites or small organic compounds. Here, the X-ray crystal structure of EaFAH, a dimeric fumarylacetoacetate hydrolase from Exiguobacterium antarcticum, was determined, and its functional properties were investigated using biochemical methods. EaFAH adopts a mixed β-sandwich roll fold with a highly flexible lid region (Val73-Leu94), and an Mg2+ ion is bound at the active site by coordinating to the three carboxylate oxygen atoms of Glu124, Glu126, and Asp155. The hydrolytic activity of EaFAH toward various substrates, including linalyl acetate was investigated using native polyacrylamide gel electrophoresis, activity staining, gel filtration, circular dichroism spectroscopy, fluorescence, and enzyme assays.

The bacterial strain Pseudomonas sp. MC1 harbors an 81-kb metabolic plasmid, which encodes enzymes involved in the conversion of naphthalene to salicylate. Of these, the enzyme NahB (cis-dihydrodiol naphthalene dehydrogenase), which catalyzes the second reaction of this pathway, binds to various substrates such as cis-1,2-dihydro-1,2-dihydroxy-naphthalene (1,2-DDN), cis-2,3-dihydro-2,3-dihydroxybiphenyl (2,3-DDB), and 3,4-dihydro-3,4-dihydroxy-2,2',5,5'-tetrachlorobiphenyl (3,4-DD-2,2',5-5-TCB). However, the mechanism underlying its broad substrate specificity is unclear owing to the lack of structural information. Here, we determined the first crystal structures of NahB in the absence and presence of NAD+ and 2,3-dihydroxybiphenyl (2,3-DB). Structure analysis suggests that the flexible substrate-binding loop allows NahB to accommodate diverse substrates. Furthermore, we defined the initial steps of substrate recognition and identified the early substrate-binding site in the substrate recognition process through the complex structure with ligands.