Evidence That Proline Focuses Movement of the Floppy Loop of Arylalkylamine N-Acetyltransferase (EC 2.3.1.87)

Abstract

Arylalkylamine N-acetyltransferase (AANAT) catalyzes the N-acetylation of serotonin, the penultimate step in the synthesis of melatonin. Pineal AANAT activity increases at night in all vertebrates, resulting in increased melatonin production. This increases circulating levels of melatonin, thereby providing a hormonal signal of darkness. Kinetic and structural analysis of AANAT has determined that one element is floppy. This element, termed Loop 1, is one of three loops that comprise the arylalkylamine binding pocket. During the course of chordate evolution, Loop 1 acquired the tripeptide CPL, and the enzyme became highly active. Here we focused on the functional importance of the CPL tripeptide and found that activity was markedly reduced when it was absent. Moreover, increasing the local flexibility of this tripeptide region by P64G and P64A mutations had the counterintuitive effect of reducing activity and reducing the overall movement of Loop 1, as estimated from Langevin dynamics simulations. Binding studies indicate that these mutations increased the off-rate constant of a model substrate without altering the dissociation constant. The structural kink and local rigidity imposed by Pro-64 may enhance activity by favoring configurations of Loop 1 that facilitate catalysis and do not become immobilized by intramolecular interactions.

Unique arylalkylamine N-acetyltransferase-2 polymorphism in Salmonids and profound variations in thermal stability and catalytic efficiency conferred by two residues

“…These results should be interpreted with care; however, differences in the ionic interaction between Glu191 and Arg104 are the sort of changes conferred by the Val160Ala mutation that could contribute to rendering saAANAT2_IA and mutant VA more thermal tolerant. It has been shown that a single or a few amino-acid substitutions can affect the impact of temperature on enzyme kinetics when these appear in regions likely to influence the conformational mobility of regions involved in catalytic conformational changes (Holland et al, 1997;Pavlicek et al, 2008). However, the differences in thermal tolerance existing between saAANAT2_IA and the VA mutant on the one hand, and saAANAT2_VV and omAANAT2_IV on the other hand, suggest that the 114 position also plays a role.…”

Drastic neofunctionalization associated with evolution of the timezyme AANAT 500 Mya

“…Activity was 5-to 10-fold higher in the retina than Major structural differences between NV-and VT-AANAT include the addition of PKA/14-3-3 binding sites flanking the core of the enzyme, which mediate regulation, the addition of histidines to facilitate catalysis by promoting removal of protons generated during the transfer of the acetyl group, and the addition of a short peptide sequence (Cys-Pro-Leu), which creates a floppy loop that facilitates arylalkylamine binding and product release in the active site. The highly conserved lysine found in the N-terminal flanking regions of VT-AANAT is thought to facilitate regulation of proteasomal proteolysis by serving as a ubiquitination site (10,13,14,25,26,28,29). Modified from ref.…”

“…The encoded proteins (206 and 207 aa) exhibited the two defining features of the GCN5 N-acetyltransferase superfamily, motifs A and B (SI Appendix, Fig. S1) (9,(22)(23)(24)(25)(26)(27)(28)(29). In addition, the sequences exhibited defining characteristics of the VT-AANATs of bony vertebrates, including motifs C and D, PKA sites nested in 14-3-3 binding domains, a Cys-ProLeu peptide sequence in a floppy loop within the binding region, and closely positioned histidine residues in the active site.…”

Dissecting the Metal Selectivity of MerR Monovalent Metal Ion Sensors in Salmonella

“…Also, the side chain of proline can wrap around to form a covalent interaction with the backbone, restricting its flexibility and limiting the conformation of neighboring residues (40). Therefore, proline has a unique role in determining local conformation and movement freedom in the folded protein, functioning as a molecular switch in the regulation of a number of cellular processes (39,41,42). In the metal sensors, the restricted set of conformations adopted by Pro at position 113 or 118 may differentially affect chain dynamics and the range of conformations the metal-binding loop can assume.…”