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Biochemistry II
Prof. Dr. Reinhard Sterner
Enzyme evolution
His Hf
Multi-enzyme complexes


Enzyme engineering


Technical advances during the last decade have enabled researchers to modify at will the properties of many naturally occurring enzymes. These approaches have also facilitated the generation of stabilised enzymes with increased turnover numbers and altered substrate- and stereo-selectivities to be used in industrial processes. Up to now, the most impressive results in enzyme design have been obtained by directed evolution. In this two-step approach random mutagenesis is used to create large enzyme repertoires, from which optimised variants are then isolated using either selection or screening techniques. In contrast to directed evolution, the alternative approach of rational enzyme design requires a detailed knowledge of a specific enzyme structure and catalytic mechanism. Although occasionally successful, rational design approaches often fail, due to a limited understanding of the subtle interplay among amino acid side chains within an enzyme active site. Recent results suggest that this bottleneck toward the acquisition of tailored enzymes can be overcome by applying sophisticated computational methods.

We have used directed evolution to establish the activity of phosphoriboslyanthranilate isomerase (TrpF) on the natural (βα)8-barrel scaffolds of ProFAR isomerase (HisA) and imidazoleglycerol phosphate synthase (HisF). Since only few mutations were necessary for the activity switch, our results suggest that TrpF, HisA and HisF have evolved from a common ancestor by a series of gene duplication and fusion events.

Moreover, we have used random mutagenesis and selection in vivo to increase the low catalytic activity of a thermostable dimeric anthranilate phosphoribosyl transferase at room temperature. The turnover number of the best mutant was increased 40-fold compared to the wild-type enzyme, due to an increased product release rate. Moreover, we have used rational design to generate a fully active monomeric variant of this enzyme by replacing hydrophobic residues at the protein interface with negatively charged ones.

The artificial control of enzymes by light is a rapidly emerging field of protein design. Along these lines, the group of Prof. Burkhard König has synthesized photoswitchable molecules that act as competitive inhibitors of Pri A, a homologue of HisA. The affinity of these photowitches for PriA is modified by irradiation, which allowed us to control the activity of PriA by light.

Enzyme evolution


It is plausible to assume that the highly efficient and specific contemporary enzymes have evolved from less sophisticated precursors.

We are studying this process for the (βα)8-barrels enzymes ProFAR isomerase (HisA) and imidazole glycerol phosphate synthase (HisF). They possess a striking internal two-fold symmetry. The pairs of N-terminal halves (designated HisA-N and HisF-N), which consist of the first four (βα) units, and the pairs of C-terminal halves (designated HisA-C and HisF-C), which consist of the last four (βα) units, display sequence identities between 16 and 26 % and rmsd values of their main-chain non-hydrogen atoms between 1.4 and 2.1 Å. When produced separately, the half-barrels HisF-N and HisF-C are homodimeric proteins with native secondary and tertiary structures, but without measurable catalytic activity. When co-expressed in vivo or refolded together in vitro, the two proteins assemble to a catalytically fully active HisF-NC heterodimer. It was concluded that both HisF and HisA are composed of two structural domains, namely the corresponding N- and C-terminal half-barrels.
These results suggest an evolutionary scenario according to which a primordial gene encoding a (βα)4-half-barrel as subunit of a homodimeric enzyme was duplicated and fused to yield a monomeric, ancestral (βα)8-barrel, which might have been the precursor of contemporary (βα)8-barrel enzymes. These postulated evolutionary events were reconstructed experimentally by generating new (βα)8-barrels from existing (βα)4-half-barrels. To this end, HisF-C was duplicated, fused and optimised to yield the stable and monomeric HisF-C***C barrel, whose X-ray structure could be solved at high resolution. Moreover, the N- and C-terminal half-barrels of HisA and HisF were fused crosswise to yield the chimeric HisAF and HisFA proteins. Using a combination of random mutagenesis and selection in vivo, high catalytic activity was established on the HisAF scaffold. The results show that stable and catalytically active (βα)8-barrels can be assembled in the laboratory by fusing, mixing and matching of (βα)4-half-barrels. Similar events might have occurred in the course of natural evolution.

Moreover, we have characterized a HisF enzyme from the last universal common ancestor of all organisms (LUCA) whose sequence was computationally reconstructed by the group of Prof. Rainer Merkl. LUCA-HisF, which contains all structural elements of modern HisF enzymes, is highly thermostable and enzymatically active. The reconstructed enzyme did, however, not catalyze the reactions of the related HisA and TrpF enzymes. This finding indicates that LUCA contained highly specific biocatalysts and thus contradicts the popular hypothesis that ancient enzymes were promiscuous.

It is generally assumed that enzymes from secondary metabolism have evolved from enzymes of primary metabolism. We have tested this hypothesis with the chorismate-utilizing enzymes anthranilate synthase (AS) from primary and isochorismate synthase (ICS) from secondary metabolism. Both enzymes catalyze mechanistically related reactions using ammonia and water as nucleophiles, respectively. We have found that the nucleophile specificity of AS can be extended from ammonia to water by just two amino acid exchanges in a channel  leading to the active site. The observed ICS/AS bi-functionality demonstrates that a secondary metabolic enyme can readily evolve from a primary metabolic enzyme without requiring an initial gene duplication event. In a general sense, our findings add to the understanding how nature has used the strucurally predetermined features of enzyme superfamilies to evolve new reactions.                                            

Multi-enzyme complexes

His Hf

Glutamine amidotransferases are a family of enzymes, whose members contain two active sites that are often located on two different polypeptide chains. The glutaminase subunit hydrolyses glutamine to glutamate and ammonia, which is added to a specific acceptor substrate at the active site of the synthase subunit. There are two important questions regarding the mechanism of glutamine amidotransferases: How are the activities at the two active sites coordinated? How is nascent ammonia transferred from the glutaminase to the synthase subunit without getting in contact with bulk water?

We are investigating these questions using imidazole glycerol phosphate (ImGP) synthase from Thermotoga maritima, which is a key metabolic enzyme that links histidine and purine biosynthesis. ImGP synthase consists of a complex of the glutaminase subunit HisH and the synthase subunit HisF, which has the (βα)8-barrel fold. A combination of biochemical investigations and X-ray crystallography revealed that glutamine hydrolysis at HisH is induced by ammonia consumption at the active site of HisF to which it migrates through a long bi-partite channel that comprises the interior of the β-barrel of HisF.

We have also analyzed a novel type of tryptophan synthase (TS) complex, which is mainly found in the phylogenetic kingdom of the Archaea. The conventional TS synthase as existing in most Bacteria and plants is composed of α- und β-subunits that form a permanent αββα comlex. In contrast, the interaction between the a- and β-subunits of the newly discovered TS complex is transient, and its stoichiometry is αββ. Moreover, the substrate of the β-subunit is not serine but phosphoserine.


Prof. Dr. Reinhard Sterner 30 15 E3_1.313 Email
Claudia Pauer (secretary) 30 04 E3_1.311 Email
Dr. Bettina Rohweder 16 39 E3_1.307 Email
Dr. Sandra Schlee 33 39 E3_1.303 Email
Dr. Andrea Kneuttinger 16 39 E3_1.307 Email
Klaus-Jürgen Tiefenbach 33 46 E3_1.309 Email
technical assistants
Christiane Endres 33 46 E3_1.309 Email
Sonja Fuchs 30 41 E3_1.305 Email
Sabine Laberer 16 39 E3_1.307 Email
Ulrike Stöckl 30 41 E3_1.305 Email
Jeannette Ueckert 33 46 E3_1.309 Email
doctoral students
Markus Busch 33 39 E3_1.303 Email
Enrico Hupfeld 33 39 E3_1.303 Email
Wolfgang Kaiser 30 41 E3_1.305 Email
Mona Linde 30 41 E3_1.305 Email
Michael Schupfner 33 39 E3_1.303 Email
Florian Semmelmann 16 39 E3_1.307 Email
MASTER students
Michael Bernhard 30 28 E3_1.317 Email
Thomas Klein 33 39 E3_1.303 Email
Cosimo Kropp 30 28 E3_1.317 Email
Fabian Ruperti 16 39 E3_1.307 Email
Martin Winter 16 39 E3_1.307 Email





My team is offering lectures, seminars, and practicals related to biochemistry.

This is a link to our courses as detailed by the course catalogue of the University of Regensburg.

All materials can be found at the E-Learning server of the University of Regensburg.


An up-to-date compilation of my publications can be found in PubMed:                        R. Sterner


Prof. Dr. Reinhard Sterner

Institute of  Biophysics and Physical Biochemistry
Room Nr. E3_1.313


University of Regensburg
Universitätsstrasse 31
93053 Regensburg

Postal Address:
University of Regensburg
93040 Regensburg

Claudia Pauer
Room Nr. E3_1.311
Phone +49-941-943 3004
Fax +49-941-943 2813

  1. Fakultät für Biologie und Vorklinische Medizin
  2. Faculty Research

Enzym evolution and protein design


Reinhard Sterner 2017 C