Structural redesign of lipase B from Candida antarctica by circular permutation and incremental truncation.
Circular permutation of Candida antarctica lipase B yields several enzyme variants with substantially increased catalytic activity. To better understand the structural and functional consequences of protein termini reorganization, we have applied protein engineering and x-ray crystallography to cp283, one of the most active hydrolase variants. Our initial investigation has focused on the role of an extended surface loop, created by linking the native N- and C-termini, on protein integrity. Incremental truncation of the loop partially compensates for observed losses in secondary structure and the permutants' temperature of unfolding. Unexpectedly, the improvements are accompanied by quaternary-structure changes from monomer to dimer. The crystal structures of one truncated variant (cp283 Delta 7) in the apo-form determined at 1.49 A resolution and with a bound phosphonate inhibitor at 1.69 A resolution confirmed the formation of a homodimer by swapping of the enzyme's 35-residue N-terminal region. Separately, the new protein termini at amino acid positions 282/283 convert the narrow access tunnel to the catalytic triad into a broad crevice for accelerated substrate entry and product exit while preserving the native active-site topology for optimal catalytic turnover.
Qian
Zhen
Z
Department of Chemistry, Emory University, 1515 Dickey Drive, Atlanta, GA 30322, USA.
Horton
John R
JR
Cheng
Xiaodong
X
Lutz
Stefan
S
D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology*; D018360:Crystallography, X-Ray; D008049:Lipase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary
D008049:Lipase
J Mol Biol. 1988 Nov 5;204(1):155-64
3216390
Curr Opin Biotechnol. 2002 Aug;13(4):390-7
12323363
Chembiochem. 2001 Oct 1;2(10):766-70
11948859
Structure. 1994 Apr 15;2(4):293-308
8087556
Nat Struct Biol. 2002 Nov;9(11):818-22
12368899
Adv Protein Chem. 1997;50:61-122
9338079
Protein Sci. 1998 Apr;7(4):848-59
9568892
Protein Sci. 1996 Jul;5(7):1290-300
8819162
Proteins. 1998 Feb 1;30(2):155-67
9489923
Trends Biotechnol. 2002 Oct;20(10):433-7
12220906
Chembiochem. 2007 Nov 5;8(16):1989-96
17876754
J Am Chem Soc. 2005 Oct 5;127(39):13466-7
16190688
Methods Mol Biol. 2003;231:143-51
12824611
Nat Struct Biol. 2000 Jul;7(7):580-5
10876245
Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
9757107
J Microbiol Biotechnol. 2007 Aug;17(8):1308-15
18051599
Mol Biol Evol. 2006 Apr;23(4):734-43
16431849
Biomol Eng. 2001 Aug;18(1):13-22
11429309
BMC Struct Biol. 2008 Feb 06;8:9
18254946
Nat Struct Biol. 1995 Sep;2(9):746-51
7552745
Protein Eng Des Sel. 2004 Feb;17(2):133-40
15047909
J Struct Biol. 2001 May-Jun;134(2-3):167-85
11551177
Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11241-6
10500161
Acta Crystallogr D Biol Crystallogr. 2002 Dec;58(Pt 12):2018-23
12454459
Nat Struct Biol. 2003 May;10(5):349-56
12679808
J Mol Biol. 1995 Apr 7;247(4):670-81
7723022
Nucleic Acids Res. 2001 Feb 15;29(4):E16
11160936
J Mol Biol. 2007 Sep 21;372(3):774-97
17681537
Acta Crystallogr A. 1991 Mar 1;47 ( Pt 2):110-9
2025413
Biochemistry. 1995 Dec 26;34(51):16838-51
8527460
Chembiochem. 2005 Jun;6(6):1051-6
15883973
Chembiochem. 2009 Feb 13;10(3):520-7
19156649
Protein Sci. 2001 Sep;10(9):1769-74
11514667
Nat Genet. 2006 Feb;38(2):168-74
16415885
Methods Enzymol. 1997;276:594-611
9048382
FASEB J. 1995 Jan;9(1):73-80
7821762
Protein Eng Des Sel. 2005 Aug;18(8):359-64
16043448
Fold Des. 1997;2(1):67-75
9080200
Biochemistry. 1997 Jul 22;36(29):8767-74
9220963
J Mol Evol. 1999 Jul;49(1):161-4
10368444
Acta Crystallogr D Biol Crystallogr. 2004 Feb;60(Pt 2):304-9
14747707
Protein Eng. 2003 Aug;16(8):599-605
12968077
J Mol Biol. 2006 Mar 3;356(4):993-1004
16405909
Annu Rev Biochem. 2005;74:433-80
15952894
J Mol Biol. 1999 Mar 5;286(4):1197-215
10047491
Biotechnol Bioeng. 2009 Apr 1;102(5):1298-315
19215031
J Mol Biol. 2005 Oct 28;353(3):719-29
16182309
Biochemistry. 1993 Nov 23;32(46):12311-8
8241117