The essential tyrosine-containing loop conformation and the role of the C-terminal multi-helix region in eukaryotic phenylalanine ammonia-lyases.
Besides the post-translationally cyclizing catalytic Ala-Ser-Gly triad, Tyr110 and its equivalents are of the most conserved residues in the active site of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), histidine ammonia-lyase (HAL, EC 4.3.1.3) and other related enzymes. The Tyr110Phe mutation results in the most pronounced inactivation of PAL indicating the importance of this residue. The recently published X-ray structures of PAL revealed that the Tyr110-loop was either missing (for Rhodospridium toruloides) or far from the active site (for Petroselinum crispum). In bacterial HAL ( approximately 500 amino acids) and plant and fungal PALs ( approximately 710 amino acids), a core PAL/HAL domain ( approximately 480 amino acids) with >or= 30% sequence identity along the different species is common. In plant and fungal PAL a approximately 100-residue long C-terminal multi-helix domain is present. The ancestor bacterial HAL is thermostable and, in all of its known X-ray structures, a Tyr83-loop-in arrangement has been found. Based on the HAL structures, a Tyr110-loop-in conformation of the P. crispum PAL structure was constructed by partial homology modeling, and the static and dynamic behavior of the loop-in/loop-out structures were compared. To study the role of the C-terminal multi-helix domain, Tyr-loop-in/loop-out model structures of two bacterial PALs (Streptomyces maritimus, 523 amino acids and Photorhabdus luminescens, 532 amino acids) lacking this C-terminal domain were also built. Molecular dynamics studies indicated that the Tyr-loop-in conformation was more rigid without the C-terminal multi-helix domain. On this basis it is hypothesized that a role of this C-terminal extension is to decrease the lifetime of eukaryotic PAL by destabilization, which might be important for the rapid responses in the regulation of phenylpropanoid biosynthesis.
Pilbák
Sarolta
S
Institute for Organic Chemistry and Research Group for Alkaloid Chemistry, Budapest University of Technology and Economics, Hungary.
Tomin
Anna
A
Rétey
János
J
Poppe
László
L
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D005057:Eukaryotic Cells; D006638:Histidine Ammonia-Lyase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D028528:Petroselinum / Q000201:enzymology / Q000235:genetics; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020637:Photorhabdus / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology / Q000235:genetics; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry
D014443:Tyrosine; D010650:Phenylalanine Ammonia-Lyase; D006638:Histidine Ammonia-Lyase