Modulating the pH-Activity Profiles of Phenylalanine Ammonia-Lyasefrom Anabaena variabilisby Modification of Center-near Surface Residues

Fan Zhang1, Nan Huang1, Li Zhou1, Wenjing Cui1, Zhongmei Liu1, Longbao Zhu2, Yi Liu3, Zhemin Zhou[1]*

Supplementary Information

Tables

SupplementaryTable1 Oligonucleotide primers used in this study

Primers / Sequence (5′→3′) a
E75N-F / AATAATGCTGTTGAATCTGGGAACCCAATTTATGGA
E75N-R / AGATGTCACTCCATAAATTGGGTTCCCAGATTCAAC
E75L-F / AATAATGCTGTTGAATCTGGGCTCCCAATTTATGGA
E75L-R / AGATGTCACTCCATAAATTGGGAGCCCAGATTCAAC
E75Q-F / AATAATGCTGTTGAATCTGGGCAGCCAATTTATGGA
E75Q-R / AGATGTCACTCCATAAATTGGCTGCCCAGATTCAAC
E75A-F / AATAATGCTGTTGAATCTGGGGCTCCAATTTATGGA
E75A-R / AGATGTCACTCCATAAATTGGAGCCCCAGATTCAAC
E75C-F / AATAATGCTGTTGAATCTGGGTGCCCAATTTATGGA
E75C-R / AGATGTCACTCCATAAATTGGGCACCCAGATTCAAC
E75D-F / AATAATGCTGTTGAATCTGGGGACCCAATTTATGGA
E75D-R / AGATGTCACTCCATAAATTGGGTCCCCAGATTCAAC
E75F-F / AATAATGCTGTTGAATCTGGGTTCCCAATTTATGGA
E75F-R / AGATGTCACTCCATAAATTGGGAACCCAGATTCAAC
E75G-F / AATAATGCTGTTGAATCTGGGGGTCCAATTTATGGA
E75G-R / AGATGTCACTCCATAAATTGGACCCCCAGATTCAAC
E75H-F / AATAATGCTGTTGAATCTGGGCATCCAATTTATGGA
E75H-R / AGATGTCACTCCATAAATTGGATGCCCAGATTCAAC
E75I-F / AATAATGCTGTTGAATCTGGGATTCCAATTTATGGA
E75I-R / AGATGTCACTCCATAAATTGGAATCCCAGATTCAAC
E75K-F / AATAATGCTGTTGAATCTGGGAAGCCAATTTATGGA
E75K-R / AGATGTCACTCCATAAATTGGCTTCCCAGATTCAAC
E75M-F / AATAATGCTGTTGAATCTGGGATGCCAATTTATGGA
E75M-R / AGATGTCACTCCATAAATTGGCATCCCAGATTCAAC
E75P-F / AATAATGCTGTTGAATCTGGGCCACCAATTTATGGA
E75P-R / AGATGTCACTCCATAAATTGGTGGCCCAGATTCAAC
E75R-F / AATAATGCTGTTGAATCTGGGCGCCCAATTTATGGA
E75R-R / AGATGTCACTCCATAAATTGGGCGCCCAGATTCAAC
E75S-F / AATAATGCTGTTGAATCTGGGTCTCCAATTTATGGA
E75S-R / AGATGTCACTCCATAAATTGGAGACCCAGATTCAAC
E75T-F / AATAATGCTGTTGAATCTGGGACCCCAATTTATGGA
E75T-R / AGATGTCACTCCATAAATTGGGGTCCCAGATTCAAC
E75V-F / AATAATGCTGTTGAATCTGGGGTTCCAATTTATGGA
E75V-R / AGATGTCACTCCATAAATTGGAACCCCAGATTCAAC
E75W-F / AATAATGCTGTTGAATCTGGGTGGCCAATTTATGGA
E75W-R / AGATGTCACTCCATAAATTGGCCACCCAGATTCAAC
E75Y-F / AATAATGCTGTTGAATCTGGGTATCCAATTTATGGAT
E75Y-R / AGATGTCACTCCATAAATTGGATACCCAGATTCAAC
N69R-F / GCATCTTGTGATTACATTAATCGCGCTGTTGAATCT
N69R-R / TGGTTCCCCAGATTCAACAGCGCGATTAATGTAATC
N69Q-F / GCATCTTGTGATTACATTAATCAGGCTGTTGAATCT
N69Q-R / TGGTTCCCCAGATTCAACAGCCTGATTAATGTAATC
N69D-F / GCATCTTGTGATTACATTAATGACGCTGTTGAATCT
N69D-R / TGGTTCCCCAGATTCAACAGCGTCATTAATGTAATCC
E72K-F / GATTACATTAATAATGCTGTTAAGTCTGGGGAACCA
E72K-R / TCCATAAATTGGTTCCCCAGACTTAACAGCATTATT
E72Q-F / GATTACATTAATAATGCTGTTCAGTCTGGGGAACCA
E72Q-R / TCCATAAATTGGTTCCCCAGACTGAACAGCATTATT
E72D-F / GATTACATTAATAATGCTGTTGACTCTGGGGAACCA
E72D-R / TCCATAAATTGGTTCCCCAGAGTCAACAGCATTATT
N89Q-F / TCTGGTTTTGGCGGTATGGCCCAGGTTGCCATATCC
N89Q-R / TTGTTCACGGGATATGGCAACCTGGGCCATACCGCC
N89D-F / TCTGGTTTTGGCGGTATGGCCGACGTTGCCATATCC
N89D-R / TTGTTCACGGGATATGGCAACGTCGGCCATACCGCC
N89H-F / TCTGGTTTTGGCGGTATGGCCATCGTTGCCATATCC
N89H-R / TTGTTCACGGGATATGGCAACGATGGCCATACCGCC
V90R-F / GGTTTTGGCGGTATGGCCAATCGCGCCATATCCCGT
V90R-R / TGCTTGTTCACGGGATATGGCGCGATTGGCCATACC
V90Q-F / GGTTTTGGCGGTATGGCCAATCAGGCCATATCCCGT
V90Q-R / TGCTTGTTCACGGGATATGGCCTGATTGGCCATACC
V90E-F / GGTTTTGGCGGTATGGCCAATGAAGCCATATCCCGT
V90E-R / TGCTTGTTCACGGGATATGGCTTCATTGGCCATACC

aBold letters denote the mutation codon.

Supplementary Table2The molar extinction coefficient for trans-cinnamate at the various pH vales

Buffer / 6.0 / 6.5 / 7.01 / 7.02 / 7.5 / 8.0 / 8.5 / 9.0 / 9.5
e / 9373 / 9386 / 9396 / 9538 / 9578 / 9632 / 9696 / 9753 / 9830

“e” is the abbreviation of the molar extinction. pH 6.0-7.01, 100 mM KH2CO3-K2HCO3 buffer; pH 7.02-9.5, 100 mM Tris-HCl buffer. The molar extinction coefficient changed slightly at the various pH vales and the difference was ignored in calculating activities.

SupplementaryTable3Surface-exposure calculation of Av-PAL by GETAREA

Residue / Total / Apolar / Backbone / Sidechain / Ratio
Asn69 / 37.33 / 11.72 / 0.46 / 36.88 / 32.30
Ala70 / 2.38 / 2.38 / 1.18 / 1.20 / 1.90
Val71 / 8.03 / 8.03 / 0.00 / 8.03 / 6.60
Glu72 / 104.22 / 41.19 / 6.97 / 97.25 / 68.90
Ser73 / 65.22 / 30.06 / 37.80 / 27.43 / 35.40
Gly74 / 40.00 / 21.42 / 40.00 / 0.00 / 45.90
Glu75 / 86.67 / 31.74 / 1.36 / 85.31 / 60.40
Pro76 / 31.52 / 29.24 / 2.28 / 29.24 / 27.80
Ile77 / 3.98 / 3.98 / 0.00 / 3.98 / 2.70
Try78 / 21.41 / 8.23 / 0.00 / 21.41 / 11.10
Gly79 / 0.00 / 0.00 / 0.00 / 0.00 / 0.00
Val80 / 0.00 / 0.00 / 0.00 / 0.00 / 0.00
Thr81 / 1.61 / 1.61 / 0.00 / 1.61 / 1.50
Ser82 / 0.03 / 0.03 / 0.00 / 0.03 / 0.00
Gly83 / 11.40 / 11.40 / 11.40 / 0.00 / 11.31
Phe84 / 42.72 / 23.47 / 19.26 / 23.46 / 13.00
Gly85 / 12.49 / 10.63 / 12.49 / 0.00 / 14.30
Gly86 / 19.68 / 6.13 / 19.68 / 0.00 / 22.60
Met87 / 26.64 / 4.61 / 25.88 / 1.03 / 19.37
Ala88 / 4.68 / 1.32 / 3.35 / 1.32 / 2.00
Asn89 / 45.73 / 3.13 / 4.84 / 40.89 / 35.80
Val90 / 93.67 / 93.65 / 4.05 / 89.62 / 73.30

Figure legend

SupplementaryFigure 1 pH-activity profiles of Asn89 and Val90 mutants.

(A) The pH optima of mutants in which Asn89 was replaced with various residues. (B) The pH optima of mutants in which Val90 was replaced with various residues. The pH optimum of each enzyme was determined by assessing the specific activity at different pH conditions (from pH 6 to 9.5). Data points correspond to the mean values of three independent experiments.

Zhemin Zhou

* To whom correspondence should be addressed.

1 Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.

2 School of Biological and Chemical Engineering, Anhui Polytechnic University, Wuhu 241000, China.

3 Key Laboratory of Food and Biotechnology, School of Food and Biotechnology, Xihua University, Chengdu 610039, China.