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.
Institute for Organic Chemistry and Research Group for Alkaloid Chemistry, Budapest University of Technology and Economics, Hungary.
Sarolta
S
Pilbák
Anna
A
Tomin
János
J
Rétey
László
L
Poppe
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