PMID- 15184119
OWN - NLM
STAT- MEDLINE
DCOM- 20040810
LR  - 20191210
IS  - 0099-2240 (Print)
IS  - 1098-5336 (Electronic)
IS  - 0099-2240 (Linking)
VI  - 70
IP  - 6
DP  - 2004 Jun
TI  - Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri 
      OX1 for synthesizing 4-methylresorcinol, methylhydroquinone, and pyrogallol.
PG  - 3253-62
AB  - Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes 
      toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this 
      study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene 
      (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives 
      of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO 
      (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, 
      T201, and F205 were used to generate random mutants. The mutants were initially 
      identified by screening with a rapid agar plate assay and then were examined 
      further by high-performance liquid chromatography and gas chromatography. Several 
      regiospecific mutants with high rates of activity were identified; for example, 
      Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation 
      mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 
      3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 
      nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 
      3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q 
      saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed 
      methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 
      3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G 
      mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 
      nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from 
      phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). 
      Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 
      0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 
      nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further 
      oxidized to trihydroxybenzenes with different regiospecificities; for example, 
      the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 
      1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene 
      was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% 
      m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% 
      m-cresol, and 47% p-cresol.
FAU - Vardar, Gonul
AU  - Vardar G
AD  - Department of Chemical Engineering, University of Connecticut, Storrs, 
      06269-3222, USA.
FAU - Wood, Thomas K
AU  - Wood TK
LA  - eng
PT  - Evaluation Study
PT  - Journal Article
PT  - Research Support, U.S. Gov't, Non-P.H.S.
PL  - United States
TA  - Appl Environ Microbiol
JT  - Applied and environmental microbiology
JID - 7605801
RN  - 0 (Culture Media)
RN  - 0 (Hydroquinones)
RN  - 0 (Resorcinols)
RN  - 01Y4A2QXY0 (Pyrogallol)
RN  - 332W51E0OC (2-methyl-1,4-hydroquinone)
RN  - EC 1.- (Mixed Function Oxygenases)
RN  - EC 1.13.- (Oxygenases)
RN  - EC 1.14.13.- (toluene 2-xylene monooxygenase)
SB  - IM
MH  - Binding Sites
MH  - Culture Media
MH  - DNA Shuffling
MH  - Escherichia coli/enzymology/genetics
MH  - Hydroquinones/*metabolism
MH  - Mixed Function Oxygenases/metabolism
MH  - Models, Molecular
MH  - Mutagenesis
MH  - Mutation
MH  - Oxygenases/*genetics/metabolism
MH  - Protein Engineering/*methods
MH  - Pseudomonas stutzeri/*enzymology/genetics
MH  - Pyrogallol/*metabolism
MH  - Resorcinols/*metabolism
MH  - Sequence Analysis, DNA
PMC - PMC427803
EDAT- 2004/06/09 05:00
MHDA- 2004/08/11 05:00
PMCR- 2004/06/01
CRDT- 2004/06/09 05:00
PHST- 2004/06/09 05:00 [pubmed]
PHST- 2004/08/11 05:00 [medline]
PHST- 2004/06/09 05:00 [entrez]
PHST- 2004/06/01 00:00 [pmc-release]
AID - 70/6/3253 [pii]
AID - 2474-03 [pii]
AID - 10.1128/AEM.70.6.3253-3262.2004 [doi]
PST - ppublish
SO  - Appl Environ Microbiol. 2004 Jun;70(6):3253-62. doi: 
      10.1128/AEM.70.6.3253-3262.2004.