Structural insights into the catalytic mechanism of aldehyde-deformylating oxygenases

The fatty alk(a/e)ne biosynthesis pathway found in cyanobacteria gained tremendous attention in recent years as a promising alternative approach for biofuel production. Cyanobacterial aldehyde-deformylating oxygenase (cADO), which catalyzes the conversion of Cn fatty aldehyde to its corresponding Cn-1 alk(a/e)ne, is a key enzyme in that pathway. Due to its low activity, alk(a/e)ne production by cADO is an inefficient process. Previous biochemical and structural investigations of cADO have provided some information on its catalytic reaction. However, the details of its catalytic processes remain unclear. Here we report five crystal structures of cADO from the Synechococcus elongates strain PCC7942 in both its iron-free and iron-bound forms, representing different states during its catalytic process. Structural comparisons and functional enzyme assays indicate that Glu144, one of the iron-coordinating residues, plays a vital role in the catalytic reaction of cADO. Moreover, the helix where Glu144 resides exhibits two distinct conformations that correlates with the different binding states of the di-iron center in cADO structures. Therefore, our results provide a structural explanation for the highly labile feature of cADO di-iron center, which we proposed to be related to its low enzymatic activity. On the basis of our structural and biochemical data, a possible catalytic process of cADO was proposed, which could aid the design of cADO with improved activity.

Structure of the catalytic domain of a state transition kinase homolog from Micromonas algae

Under natural environments, plants and algae have evolved various photosynthetic acclimation mechanisms in response to the constantly changing light conditions. The state transition and long-term response processes in photosynthetic acclimation involve remodeling and composition alteration of thylakoid membrane. A chloroplast protein kinase named Stt7/STN7 has been found to have pivotal roles in both state transition and long-term response. Here we report the crystal structures of the kinase domain of a putative Stt7/STN7 homolog from Micromonas sp. RCC299 (MsStt7d) in the apo form and in complex with various nucleotide substrates. MsStt7d adopts a canonical protein kinase fold and contains all the essential residues at the active site. A novel hairpin motif, found to be a conserved feature of the Stt7/STN7 family and indispensable for the kinase stability, interacts with the activation loop and fixes it in an active conformation. We have also demonstrated that MsStt7d is a dualspecifi city kinase that phosphorylates both Thr and Tyr residues. Moreover, preliminary in vitro data suggest that it might be capable of phosphorylating a consensus N-terminal pentapeptide of light-harvesting proteins Micromonas Lhcp4 and Arabidopsis Lhcb1 directly. The potential peptide/protein substrate binding site is predicted based on the location of a pseudo-substrate contributed by the adjacent molecule within the crystallographic dimer. The structural and biochemical data presented here provide a framework for an improved understanding on the role of Stt7/STN7 in photosynthetic acclimation.