Improving the catalytic activity of a thermophilic enzyme at low temperatures.


Abstract

Enzymes from thermophilic organisms often are barely active at low temperatures. To obtain a better understanding of this sluggishness, we used DNA shuffling to mutagenize the trpC gene, which encodes indoleglycerol phosphate synthase, from the hyperthermophile Sulfolobus solfataricus. Mutants producing more active protein variants were selected by genetic complementation of an Escherichia coli mutant bearing a trpC deletion. Single amino acid changes and combinations of these changes improved growth appreciably. Five singly and doubly altered protein variants with changes at the N- and C-termini, or at the phosphate binding site, were purified and characterized with regard to their kinetics of enzymatic catalysis, product binding, cleavage by trypsin, and inactivation by heat. Turnover numbers of the purified variant proteins correlated with the corresponding growth rates, showing that the turnover number was the selected trait. Although the affinities for both the substrate and the product decreased appreciably in most protein variants, these defects were offset by the accumulation of high levels of the enzyme's substrate. Rapid mixing of the product indoleglycerol phosphate with the parental enzyme revealed that the enzyme's turnover number at low temperatures is limited by the dissociation of the enzyme-product complex. In contrast, representative protein variants bind and release the product far more rapidly, shifting the bottleneck to the preceding chemical step. The turnover number of the parental enzyme increases with temperature, suggesting that its structural rigidity is responsible for its poor catalytic activity at low temperatures. In support of this interpretation, the rate of trypsinolysis or of thermal denaturation is accelerated significantly in the activated protein variants. Study holds ProTherm entries: 8004, 8005, 8006, 8007, 8008, 8009, 8010, 8011, 8012, 8013, 8014, 8015, 8016, 8017, 8018, 8019, 8020, 8021, 8022, 8023, 8024, 8025, 8026, 8027, 8028, 8029, 8030, 8031, 8032, 8033 Extra Details: additive : DTT(1 mM), catalytic activity; hyperthermophile; phosphate binding site;,thermal denaturation

Submission Details

ID: j47umWpD4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:35 p.m.

Version: 1

Publication Details
Merz A;Yee MC;Szadkowski H;Pappenberger G;Crameri A;Stemmer WP;Yanofsky C;Kirschner K,Biochemistry (2000) Improving the catalytic activity of a thermophilic enzyme at low temperatures. PMID:10653631
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
5AOU 2016-08-17 1.1 Structure of the engineered retro-aldolase RA95.5-8F apo
4A29 2012-11-07 1.1 Structure of the engineered retro-aldolase RA95.0
5AN7 2016-08-17 1.1 Structure of the engineered retro-aldolase RA95.5-8F with a bound 1,3-diketone inhibitor
4OU1 2014-03-05 1.25 Crystal structure of a computationally designed retro-aldolase covalently bound to folding probe 1 [(6-methoxynaphthalen-2-yl)(oxiran-2-yl)methanol]
4A2R 2012-11-07 1.3 Structure of the engineered retro-aldolase RA95.5-5
5K7J 2016-08-03 1.39 Structure of designed zinc binding protein ZE2 bound to Zn2+
4A2S 2012-11-07 1.4 Structure of the engineered retro-aldolase RA95.5
3TC7 2011-08-24 1.5 Crystal Structure of Engineered Protein. Northeast Structural Genomics Consortium Target OR62.
3NYZ 2011-06-29 1.51 Crystal Structure of Kemp Elimination Catalyst 1A53-2
3NZ1 2011-06-29 1.56 Crystal Structure of Kemp Elimination Catalyst 1A53-2 Complexed with Transition State Analog 5-Nitro Benzotriazole
3TC6 2011-08-24 1.6 Crystal Structure of Engineered Protein. Northeast Structural Genomics Consortium Target OR63.
4LNY 2013-08-07 1.93 Crystal Structure of Engineered Protein, Northeast Structural Genomics Consortium Target OR422
1A53 1999-03-23 2.0 COMPLEX OF INDOLE-3-GLYCEROLPHOSPHATE SYNTHASE FROM SULFOLOBUS SOLFATARICUS WITH INDOLE-3-GLYCEROLPHOSPHATE AT 2.0 A RESOLUTION
1IGS 1996-07-11 2.0 INDOLE-3-GLYCEROLPHOSPHATE SYNTHASE FROM SULFOLOBUS SOLFATARICUS AT 2.0 A RESOLUTION
1JUL 1997-07-07 2.0 INDOLE-3-GLYCEROLPHOSPHATE SYNTHASE FROM SULFOLOBUS SOLFATARICUS IN A SECOND ORTHORHOMBIC CRYSTAL FORM
1LBF 2002-06-12 2.05 CRYSTAL STRUCTURE OF INDOLE-3-GLYCEROL PHOSPHATE SYNTASE (IGPS)WITH REDUCED 1-(O-CABOXYPHENYLAMINO)-1-DEOXYRIBULOSE 5-PHOSPHATE (RCDRP)
4IWW 2013-08-21 2.3 Computational Design of an Unnatural Amino Acid Metalloprotein with Atomic Level Accuracy
1LBL 2002-09-18 2.4 Crystal structure of indole-3-glycerol phosphate synthase (IGPS) in complex with 1-(o-carboxyphenylamino)-1-deoxyribulose 5'-phosphate (CdRP)
4IX0 2013-08-21 2.5 Computational Design of an Unnatural Amino Acid Metalloprotein with Atomic Level Accuracy
1JUK 1997-07-07 2.5 INDOLE-3-GLYCEROLPHOSPHATE SYNTHASE FROM SULFOLOBUS SOLFATARICUS IN A TRIGONAL CRYSTAL FORM
2C3Z 2005-10-13 2.8 Crystal structure of a truncated variant of indole-3-glycerol phosphate synthase from Sulfolobus solfataricus
6NW4 2019-07-24 3.0 Evolution of a computationally designed Kemp eliminase

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Indole-3-glycerol phosphate synthase Q06121 TRPC_SACS2