Catalytic Ability Improvement of Phenylalanine Hydroxylase from Chromobacterium violaceum by N-Terminal Truncation and Proline Introduction = Catalytic Ability Improvement of Phenylalanine Hydroxylase from Chromobacterium violaceum by N-Terminal Truncation and Proline Introduction

다국어 초록 (Multilingual Abstract)

Phenylalanine hydroxylase from Chromobacterium violaceum (CvPAH) is a monomeric enzyme that converts phenylalanine to tyrosine. It shares high amino acid identity and similar structure with a subunit of human phenylalanine hydroxylase that is a tetramer, resulting in the latent application in medications. In this study, semirational design was applied to CvPAH to improve the catalytic ability based on molecular dynamics simulation analyses. Four Nterminal truncated variants and one single point variant were constructed and characterized. The D267P variant showed a 2.1-fold increased thermal stability compared to the wild type, but lower specific activity was noted compared with the wild type. The specific activity of all truncated variants was a greater than 25% increase compared to the wild type, and these variants showed similar or slightly decreased thermostability with the exception of the N-Δ9 variant. Notably, the N-Δ9 variant exhibited a 1.2-fold increased specific activity, a 1.3-fold increased thermostability and considerably increased catalytic activity under the neutral environment compared with the wild type. These properties of the N-Δ9 variant could advance medical and pharmaceutical applications of CvPAH. Our findings indicate that the N-terminus might modulate substrate binding, and are directives for further modification and functional research of PAH and other enzymes.

참고문헌 (Reference)

1 Zhu T, "The predictive value of genetic analyses in the diagnosis of tetrahydrobiopterin (BH4)-responsiveness in Chinese phenylalanine hydroxylase deficiency patients" 7 : 6762-, 2017

2 Ge M, "The contribution of proline residues to protein stability is associated with isomerization equilibrium in both unfolded and folded states" 13 : 481-489, 2009

3 Muntau AC, "Tetrahydrobiopterin as an alternative treatment for mild phenylketonuria" 347 : 2122-2132, 2002

4 Flatmark T, "Structural insight into the aromatic amino acid hydroxylases and their disease-related mutant forms" 99 : 2137-2160, 1999

5 Erlandsen H, "Structural comparison of bacterial and human iron-dependent phenylalanine hydroxylases : similar fold, different stability and reaction rates" 320 : 645-661, 2002

6 Huang J, "Stabilization of an α/β-hydrolase by introducing proline residues : salicylic acid binding protein 2 from tobacco" 54 : 4330-4341, 2015

7 Phillips JC, "Scalable molecular dynamics with NAMD" 26 : 1781-1802, 2005

8 Zoidakis J, "Role of the second coordination sphere residue tyrosine 179 in substrate affinity and catalytic activity of phenylalanine hydroxylase" 9 : 289-296, 2004

9 Singh MK, "Role of an N-terminal extension in stability and catalytic activity of a hyperthermostable α/β hydrolase fold esterase" 30 : 559-570, 2017

10 Abu-Omar MM, "Reaction mechanisms of mononuclear non-heme iron oxygenases" 105 : 2227-2252, 2005

11 Nakata H, "Phenylalanine hydroxylase from Chromobacterium violaceum, purification and characterization" 254 : 1829-1833, 1979

12 Stojanovski BM, "Murine erythroid 5-aminolevulinate synthase : Truncation of a disordered N-terminal extension is not detrimental for catalysis" 1864 : 441-452, 2016

13 Yan M, "Molecular dynamics simulations of HIV-1 protease monomer : Assembly of N-terminus and C-terminus into bsheet in water solution" 70 : 731-738, 2008

14 Carr RT, "Mechanism of metal-independent hydroxylation by Chromobacterium violaceum phenylalanine hydroxylase" 34 : 7525-7532, 1995

15 Keil S, "Longterm follow-up and outcome of phenylketonuria patients on sapropterin : a retrospective study" 131 : e1881-e1888, 2013

16 Subedi BP, "Kinetic mechanism and intrinsic rate constants for the reaction of a bacterial phenylalanine hydroxylase" 55 : 6848-6857, 2016

17 Yew NS, "Erythrocytes encapsulated with phenylalanine hydroxylase exhibit improved pharmacokinetics and lowered plasma phenylalanine levels in normal mice" 109 : 339-344, 2013

18 Kino K, "Enhancement of L-tryptophan 5-hydroxylation activity by structure-based modification of L-phenylalanine 4-hydroxylase from Chromobacterium violaceum" 108 : 184-189, 2009

19 Tian J, "Enhanced thermostability of methyl parathion hydrolase from Ochrobactrum sp, M231 by rational engineering of a glycine to proline mutation" 277 : 4901-4908, 2010

20 Kazlauskas R, "Engineering more stable proteins" 47 : 9026-9045, 2018

21 Levy HL, "Efficacy of sapropterin dihydrochloride(tetrahydrobiopterin, 6R-BH4)for reduction of phenylalanine concentration in patients with phenylketonuria : a phase III randomised placebo-controlled study" 370 : 504-510, 2007

22 Zoidakis J, "Effect of temperature, pH, and metals on the stability and activity of phenylalanine hydroxylase from Chromobacterium violaceum" 99 : 771-775, 2005

23 Onishi A, "Cloning and expression of Chromobacterium violaceum phenylalanine hydroxylase in Escherichia coli and comparison of amino acid sequence with mammalian aromatic amino acid hydroxylases" 266 : 18454-18459, 1991

24 MacKerell AD, "All-atom empirical potential for molecular modeling and dynamics studies of proteins" 102 : 3586-3616, 1998

25 Ronau JA, "A conserved acidic residue in phenylalanine hydroxylase contributes to cofactor affinity and catalysis" 53 : 6834-6848, 2014