Isopenicillin N Synthase: Crystallographic Studies

Isopenicillin N synthase (IPNS) is a non-heme iron oxidase (NHIO) that catalyses the cyclisation of tripeptide δ-(l -α-aminoadipoyl)-l -cysteinyl-d -valine (ACV) to bicyclic isopenicillin N (IPN). Over the last 25 years, crystallography has shed considerable light on the mechanism of IPNS catalysis. The first crystal structure, for apo-IPNS with Mn bound in place of Fe at the active site, reported in 1995, was also the first structure for a member of the wider NHIO family. This was followed by the anaerobic enzyme-substrate complex IPNS−Fe−ACV (1997), this complex plus nitric oxide as a surrogate for co-substrate dioxygen (1997), and an enzyme product complex (1999). Since then, crystallography has been used to probe many aspects of the IPNS reaction mechanism, by crystallising the protein with a diversity of substrate analogues and triggering the oxidative reaction by using elevated oxygen pressures to force the gaseous co-substrate throughout protein crystals and maximise synchronicity of turnover in crystallo. In this way, X-ray structures have been elucidated for a range of complexes closely related to and/or directly derived from key intermediates in the catalytic cycle, thereby answering numerous mechanistic questions that had arisen from solution-phase experiments, and posing many new ones. The results of these crystallographic studies have, in turn, informed computational experiments that have brought further insight. These combined crystallographic and computational investigations augment and extend the results of earlier spectroscopic analyses and solution phase studies of IPNS turnover, to enrich our understanding of this important protein and the wider NHIO enzyme family.     

A cyclobutanone analogue mimics penicillin in binding to isopenicillin N synthase

A carbocyclic analogue of the beta-lactam antibiotic isopenicillin N (IPN) has been synthesised and cocrystallised with isopenicillin N synthase (IPNS), the central enzyme in the biosynthesis of penicillin antibiotics. The crystal structure of the IPNS-cyclobutanone complex reveals an active site environment similar to that seen in the enzyme-product complex generated by turnover of the natural substrate within the crystalline protein. The IPNS-cyclobutanone structure demonstrates that the product analogue is tethered to the protein by hydrogen bonding and salt bridge interactions with its carboxylate groups, as seen previously for the natural substrate and product. Furthermore, the successful cocrystallisation of this analogue with IPNS provides firm structural evidence for the utility of such cyclobutanone derivatives as hydrolytically stable analogues of bicyclic beta-lactams.     

Structural Studies on the Reaction of Isopenicillin N Synthase with a Sterically Demanding Depsipeptide Substrate Analogue

Isopenicillin N synthase (IPNS) is a nonheme iron(II)‐dependent oxidase that catalyses the central step in penicillin biosynthesis, conversion of the tripeptide δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐valine (ACV) to isopenicillin N (IPN). This report describes mechanistic studies using the analogue δ‐(L ‐α‐aminoadipoyl)‐(3S‐methyl)‐L ‐cysteine D ‐α‐hydroxyisovaleryl ester (A^SmCOV), designed to intercept the catalytic cycle at an early stage. A^SmCOV incorporates two modifications from the natural substrate: the second and third residues are joined by an ester, so this analogue lacks the key amide of ACV and cannot form a β‐lactam; and the cysteinyl residue is substituted at its β‐carbon, bearing a (3S)‐methyl group. It was anticipated that this methyl group will impinge directly on the site in which the co‐substrate dioxygen binds. The novel depsipeptide A^SmCOV was prepared in 13 steps and crystallised with IPNS anaerobically. The 1.65 Å structure of the IPNS–Fe^II–A^SmCOV complex reveals that the additional β‐methyl group is not oriented directly into the oxygen binding site, but does increase steric demand in the active site and increases disorder in the position of the isovaleryl side chain. Crystals of IPNS–Fe^II–A^SmCOV were incubated with high‐pressure oxygen gas, driving substrate turnover to a single product, an ene‐thiol/C‐hydroxylated depsipeptide. A mechanism is proposed for the reaction of A^SmCOV with IPNS, linking this result to previous crystallographic studies with related depsipeptides and solution‐phase experiments with cysteine‐methylated tripeptides. This result demonstrates that a (3S)‐methyl group at the substrate cysteinyl β‐carbon is not in itself a block to IPNS activity as previously proposed, and sheds further light on the steric complexities of IPNS catalysis.     

The Interaction of Isopenicillin N Synthase with Homologated Substrate Analogues δ‐(L‐α‐Aminoadipoyl)‐L‐homocysteinyl‐D‐Xaa Characterised by Protein Crystallography

Isopenicillin N synthase (IPNS) converts the linear tripeptide δ‐(L ‐α‐aminoadipoyl)‐L ‐cysteinyl‐D ‐valine (ACV) into bicyclic isopenicillin N (IPN) in the central step in the biosynthesis of penicillin and cephalosporin antibiotics. Solution‐phase incubation experiments have shown that IPNS turns over analogues with a diverse range of side chains in the third (valinyl) position of the substrate, but copes less well with changes in the second (cysteinyl) residue. IPNS thus converts the homologated tripeptides δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐valine (AhCV) and δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐allylglycine (AhCaG) into monocyclic hydroxy‐lactam products; this suggests that the additional methylene unit in these substrates induces conformational changes that preclude second ring closure after initial lactam formation. To investigate this and solution‐phase results with other tripeptides δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐Xaa, we have crystallised AhCV and δ‐(L ‐α‐aminoadipoyl)‐L ‐homocysteinyl‐D ‐S‐methylcysteine (AhCmC) with IPNS and solved crystal structures for the resulting complexes. The IPNS:Fe^II:AhCV complex shows diffuse electron density for several regions of the substrate, revealing considerable conformational freedom within the active site. The substrate is more clearly resolved in the IPNS:Fe^II:AhCmC complex, by virtue of thioether coordination to iron. AhCmC occupies two distinct conformations, both distorted relative to the natural substrate ACV, in order to accommodate the extra methylene group in the second residue. Attempts to turn these substrates over within crystalline IPNS using hyperbaric oxygenation give rise to product mixtures.     

Isopenicillin N synthase binds δ-(L-α-aminoadipoyl)-L-cysteinyl-D-thia-allo-isoleucine through both sulfur atoms

Isopenicillin N synthase (IPNS) catalyses the synthesis of isopenicillin N (IPN), the biosynthetic precursor to penicillin and cephalosporin antibiotics. IPNS is a non‐heme iron(II) oxidase that mediates the oxidative cyclisation of the tripeptide δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐valine (ACV) to IPN with a concomitant reduction of molecular oxygen to water. Solution‐phase incubation experiments have shown that, although IPNS can turn over analogues with a diverse range of hydrocarbon side chains in the third (valinyl) position of its substrate, the enzyme is much less tolerant of polar residues in this position. Thus, although IPNS converts δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐isoleucine (ACI) and AC‐D ‐allo‐isoleucine (ACaI) to penam products, the isosteric sulfur‐containing peptides AC‐D ‐thiaisoleucine (ACtI) and AC‐D ‐thia‐allo‐isoleucine (ACtaI) are not turned over. To determine why these peptides are not substrates, we crystallized ACtaI with IPNS. We report the synthesis of ACtaI and the crystal structure of the IPNS:Fe^II:ACtaI complex to 1.79 Å resolution. This structure reveals direct ligation of the thioether side chain to iron: the sulfide sulfur sits 2.66 Å from the metal, squarely in the oxygen binding site. This result articulates a structural basis for the failure of IPNS to turn over these substrates.     

Variations of the 2-His-1-carboxylate theme in mononuclear non-heme FeII oxygenases

A facial triad of two histidine side chains and one aspartate or glutamate side chain forms the canonical metal-coordinating motif in the catalytic centers of various mononuclear non-heme Fe(II) enzymes. Although these active sites are based on totally unrelated protein folds and bring about a wide range of chemical transformations, most of them share the ability to couple dioxygen reduction with the oxygenation of an organic substrate. With the increasing number of protein structures now solved, it has become clear that the 2-His-1-carboxylate signature is less of a paradigm for non-heme Fe(II) active sites than had long been thought and that it can be replaced by alternative metal centers in various oxygenases, the structure-function relationships and proposed catalytic mechanisms of which are reviewed here. Metal coordination through three histidines and one glutamate constitutes the classical motif described for enzyme members of the cupin protein superfamily, such as aci-reductone dioxygenase and quercetin dioxygenase, multiple metal forms of which (including the Fe(II) type) are found in nature. Cysteine dioxygenase and diketone dioxygenase, which are strictly Fe(II)-dependent oxygenases based on the cupin fold, bind the catalytic metal through the homologous triad of histidines, but lack the fourth glutamate ligand. An alpha-ketoglutarate-dependent Fe(II) halogenase shows metal coordination by two histidines as the only protein-derived ligands, whilst carotene oxygenase, from a different protein fold family, features an Fe(II) site consisting of four histidine side chains. These recently discovered metallocenters are discussed with respect to their metal-binding properties and the reaction coordinates of the O(2)-dependent conversions they catalyze.     

Oxygen utilisation by isopenicillin n synthase from penicillium chrysogenum

The enzyme isopenicillin N synthase (IPNS) converts δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N; an equimolar amount of oxygen is used in this oxidative ring closure reaction. Oxygen uptake rates of the reaction catalysed by partially purified IPNS from Penicillium chrysogenum SC 6140 and P2 were measured using an oxygen electrode. In contrast to published properties of Cephalosporium acremonium IPNS, the enzyme from P. chrysogenum was not stimulated by the addition of glutathione and showed reduced stimulation by Fe²⁺. The analysis of oxygen uptake rates showed the reaction to be first order with respect to oxygen concentration and the K_m for ACV to be 0·4 mmol dm^?3. The implications of these results for cell-free reactions using this enzyme and penicillin fermentations are discussed.     

Nitrosymmetallochelates—I. Mechanism of FeNO—EDTA complex formation and its oxidation

A nitrosyliron(II) complex with EDTA was produced by reaction of the Fe(II)—EDTA complex and nitrous acid in citric acid—phosphate buffer solution. The experimental results pointed out that nitrous acid is reduced to nitrous oxide by Fe(II)—EDTA complex followed by the formation reaction of nitrosyliron(II) EDTA complex. The composition of this complex was determined to be Fe(II) (NO)₂EDTA by both electrochemical and spectroscopic methods.A mechanism of the oxidation of Fe(II) (NO)₂EDTA was proposed in which nitrous oxide breaks away from the iron ion. The photoeffect on this reaction is discussed.     

Improved ORR activity of non-noble metal electrocatalysts by increasing ligand and metal ratio in synthetic complex precursors

In an effort to improve oxygen reduction reaction (ORR) activity by increasing the catalytic active site density in carbon-supported non-noble metal catalysts, several nitrogen-containing catalysts were synthesized through a heat treatment process at 900 °C using precursor complexes of Fe(II) and tripyridyl triazine (TPTZ). Fe to TPTZ mole ratios of 1:2, 1:3, 1:4, 1:5, 1:6, and 1:7 were used to prepare the precursor complexes. X-ray diffraction and surface electrochemical techniques were used to characterize these catalysts (Fe–N_x/C), and revealed that when the amount of TPTZ in the precursor complex was increased, the decomposition of Fe–N_x sites, which are considered active sites for the ORR, was effectively reduced, resulting in higher Fe–N_x site density and thus improving the catalysts’ ORR activity. This beneficial effect was validated through rotating disk electrode tests and analysis of the ORR kinetics catalyzed by these catalysts. The obtained results showed that as the Fe to TPTZ mole ratio in the precursor complex was decreased, the catalytic ORR activity of Fe–N_x/C increased monotonically in the mole ratio range of 1:2–1:6. Therefore, increasing the amount of ligand in the precursor metal complex was demonstrated to be an effective way to reduce the decomposition of ORR active site density and thereby enhance the ORR activity of non-noble metal catalysts.     

A Second,Druggable Binding Site in UDP‐Galactopyranose Mutase from Mycobacterium tuberculosis?

UDP‐galactopyranose mutase (UGM), a key enzyme in the biosynthesis of mycobacterial cell walls, is a potential target for the treatment of tuberculosis. In this work, we investigate binding models of a non‐substrate‐like inhibitor, MS‐208, with M. tuberculosis UGM. Initial saturation transfer difference (STD) NMR experiments indicated a lack of direct competition between MS‐208 and the enzyme substrate, and subsequent kinetic assays showed mixed inhibition. We thus hypothesized that MS‐208 binds at an allosteric binding site (A‐site) instead of the enzyme active site (S‐site). A candidate A‐site was identified in a subsequent computational study, and the overall hypothesis was supported by ensuing mutagenesis studies of the A‐site. Further molecular dynamics studies led us to propose that MS‐208 inhibition occurs by preventing complete closure of an active site mobile loop that is necessary for productive substrate binding. The results suggest the presence of an A‐site with potential druggability, opening up new opportunities for the development of novel drug candidates against tuberculosis.     

A study of the Fe(III)/Fe(II)-triethanolamine complex redox couple for redox flow battery application

The electrochemical behavior of the Fe(III)/Fe(II)-triethanolamine(TEA) complex redox couple in alkaline medium and influence of the concentration of TEA were investigated. A change of the concentration of TEA mainly produces the following two results. (1) With an increase of the concentration of TEA, the solubility of the Fe(III)-TEA can be increased to 0.6 M, and the solubility of the Fe(II)-TEA is up to 0.4 M. (2) In high concentration of TEA with the ratio of TEA to NaOH ranging from 1 to 6, side reaction peaks on the cathodic main reaction of the Fe(III)-TEA complex at low scan rate can be minimized. The electrode process of Fe(III)-TEA/Fe(II)-TEA is electrochemically reversible with higher reaction rate constant than the uncomplexed species. Constant current charge-discharge shows that applying anodic active materials of relatively high concentrations facilitates the improvement of cell performance. The open-circuit voltage of the Fe-TEA/Br₂ cell with the Fe(III)-TEA of 0.4 M, after full charging, is nearly 2.0 V and is about 32% higher than that of the all-vanadium batteries, together with the energy efficiency of approximately 70%. The preliminary exploration shows that the Fe(III)-TEA/Fe(II)-TEA couple is electrochemically promising as negative redox couple for redox flow battery (RFB) application.     

Combinatorial exploration of the catalytic site of a drug-resistant dihydrofolate reductase: creating alternative functional configurations

We have applied a global approach to enzyme active site exploration, where multiple mutations were introduced combinatorially at the active site of Type II R67 dihydrofolate reductase (R67 DHFR), creating numerous new active site environments within a constant framework. By this approach, we combinatorially modified all 16 principal amino acids that constitute the active site of this enzyme. This approach is fundamentally different from active site point mutation in that the native active site context is no longer accounted for. Among the 1536 combinatorially mutated active site variants of R67 DHFR we created, we selected and kinetically characterized three variants with highly altered active site compositions. We determined that they are of high fitness, as defined by a complex function consisting jointly of catalytic activity and resistance to trimethoprim. The k(cat) and K(M) values were similar to those for the native enzyme. The favourable Delta(DeltaG) values obtained (ranging from -0.72 to -1.08 kcal/mol) suggest that, despite their complex mutational pattern, no fundamental change in the catalytic mechanism has occurred. We illustrate that combinatorial active site mutagenesis can allow for the creation of compensatory mutations that could not be predicted and thus provides a route for more extensive exploration of functional sequence space than is allowed by point mutation.     

Fe(Ⅱ)-EDTA吸收一氧化氮动力学研究

本文提出一种用Fe(Ⅱ)-EDTA溶液同时脱除硝酸尾气中NO_x和合成氨原料气中H_2S的方法,并对Fe(Ⅱ)-EDTA溶液吸收一氧化氮动力学进行了研究.根据络合反应:NO+Fe(Ⅱ)-EDTA(?)Fe(Ⅱ)(NO)EDTA属快速(2,1)级可逆反应的特点,建立起适合于一般形式A+B(?)E的快速(2,1)级可逆反应宏观吸收动力学数学模型,并和现有的其它模型进行比较.将所导出的数学模型与所测得的Fe(Ⅱ)-EDTA吸收NO动力学实验数据拟合,采用Marquardt法搜索、选优,求得Fe(Ⅱ)-EDTA吸收NO动力学关系式.文章还提出了测定数学模型中各个物性参数的实验方法.     

Hydrazine drastically promoted Fenton oxidation of bisphenol A catalysed by a FeIII–Co Prussian blue analogue

Herein, for the first time it has been demonstrated that hydrazine (Hz) could significantly promote the bisphenol A (BPA) degradation in the Fenton reaction catalysed by Fe[Co(CN)₆]2H₂O Prussian blue analogue (Fe^III–Co PBA). Results indicate that the dramatic enhancement of BPA degradation could be partly attributed to the induced homogeneous Fenton reaction by the enhanced dissolution of Fe^III–Co PBA. Meanwhile, the Hz coordinated iron site (H₂NH₂N–Fe), which is evolved from the original water coordinated iron site (H₂O–Fe), was identified as the main active site. A possible reaction pathway involving the proposed active iron species was proposed.     

Hedycaryol Synthase in Complex with Nerolidol Reveals Terpene Cyclase Mechanism

The biosynthesis of terpenes is catalysed by class I and II terpene cyclases. Here we present structural data from a class I hedycaryol synthase in complex with nerolidol, serving as a surrogate for the reaction intermediate nerolidyl diphosphate. This prefolded ligand allows mapping of the active site and hence the identification of a key carbonyl oxygen of Val179, a highly conserved helix break (G1/2) and its corresponding helix dipole. Stabilising the carbocation at the substrate's C1 position, these elements act in concert to catalyse the 1,10 ring closure, thereby exclusively generating the anti‐Markovnikov product. The delineation of a general mechanistic scaffold was confirmed by site‐specific mutations. This work serves as a basis for understanding carbocation chemistry in enzymatic reactions and should contribute to future application of these enzymes in organic synthesis.     

Effect of cooperativity on microbial growth

The classical Monod equation for the specific growth rate of a microbial population can be derived by assuming a single substrate enzyme-catalysed reaction (Michaelis-Menten kinetics) as the rate-limiting step in microbial growth. In some cases the enzyme which catalyses the rate-limiting step in microbial growth may have more than one substrate binding site and the binding of one substrate molecule to the enzyme facilitates the binding of the next substrate molecule (cooperativity). The presence of cooperativity changes the form of the Michaelis-Menten equation for enzyme-catalysed substrate reactions and also the Monod equation for microbial growth. The number of interacting active sites on an enzyme molecule is an additional parameter in this case. In this article, the cooperative growth model for n interacting sites on the enzyme is derived and compared with the classical Monod model.     

Structure and mechanisms of Escherichia coli aspartate transcarbamoylase

Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein. Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, L-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity. In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60 ? from the active site, inducing structural alterations that modulate catalytic activity. The delineation of the structure and function in this particular model system will help in understanding the molecular basis of cooperativity and allosteric regulation in other systems as well.     

Selection of metalloenzymes by catalytic activity using phage display and catalytic elution

The metallo-beta-lactamase betaLII from Bacillus cereus 569/H/9 was displayed on the filamentous phage fd. The phage-bound enzyme fd-betaLII was shown to be active on benzylpenicillin as substrate; it could be inactivated by complexation of the essential zinc(II) ion with EDTA and reactivated by addition of a zinc(II) salt. A selection process was designed to extract active phage-bound enzymes from libraries of mutants in three steps: 1. inactivation of active phage-bound enzymes by metal ion complexation, 2. binding to substrate-coated magnetic beads, 3. release of phages capable of transforming the substrate into product upon zinc salt addition. The selection process was first successfully tested on model mixtures containing fd-betaLII plus either a dummy phage, a phage displaying an inactive mutant of the serine beta-lactamase TEM-1, or inactive and low-activity mutants of betaLII. The selection was then applied to extract active phage-bound enzymes from a library of mutants generated by mutagenic polymerase chain reaction (PCR). The activity of the library was shown to increase 60-fold after two rounds of selection. Eleven clones from the second round were randomly picked for sequencing and to characterize their activity and stability.     

Monooxygenase activity of type 3 copper proteins

The molecular mechanism of the monooxygenase (phenolase) activity of type 3 copper proteins has been examined in detail both in the model systems and in the enzymatic systems. The reaction of a side-on peroxo dicopper(II) model compound ( A) and neutral phenols proceeds via a proton-coupled electron-transfer (PCET) mechanism to generate phenoxyl radical species, which collapse each other to give the corresponding C-C coupling dimer products. In this reaction, a bis(mu-oxo)dicopper(III) complex ( B) generated by O-O bond homolysis of A is suggested to be a real active species. On the other hand, the reaction of lithium phenolates (deprotonated form of phenols) with the same side-on peroxo dicopper(II) complex proceeds via an electrophilic aromatic substitution mechanism to give the oxygenated products (catechols). The mechanistic difference between these two systems has been discussed on the basis of the Marcus theory of electron transfer and Hammett analysis. Mechanistic details of the monooxygenase activity of tyrosinase have also been examined using a simplified enzymatic reaction system to demonstrate that the enzymatic reaction mechanism is virtually the same as that of the model reaction, that is, an electrophilic aromatic substitution mechanism. In addition, the monooxygenase activity of the oxygen carrier protein hemocyanin has been explored for the first time by employing urea as an additive in the reaction system. In this case as well, the ortho-hydroxylation of phenols to catechols has been demonstrated to involve the same ionic mechanism.     

Modified substrate specificity of L-hydroxyisocaproate dehydrogenase derived from structure-based protein engineering

L-2-Hydroxyisocaproate dehydrogenase (L-HicDH) is characterized by a broad substrate specificity and utilizes a wide range of 2-oxo acids branched at the C4 atom. Modifications have been made to the sequence of the NAD(H)-dependent L-HicDH from Lactobacillus confusus in order to define and alter the region of substrate specificity towards various 2- oxocarbonic acids. All variations were based on a 3D-structure model of the enzyme using the X-ray coordinates of the functionally related L- lactate dehydrogenase (L-LDH) from dogfish as a template. This protein displays only 23% sequence identity to L-HicDH. The active site of L- HicDH was modelled by homology to the L-LDH based on the conservation of catalytically essential residues. Substitutions of the active site residues Gly234, Gly235, Phe236, Leu239 and Thr245 were made in order to identify their unique participation in substrate recognition and orientation. The kinetic properties of the L239A, L239M, L236V and T245A enzyme variants confirmed the structural model of the active site of L-HicDH. The substrates 2-oxocaproate, 2-oxoisocaproate, phenylpyruvate, phenylglyoxylate, keto-tert-leucine and pyruvate were fitted into the active site of the subsequently refined model. In order to design dehydrogenases with an improved substrate specificity towards keto acids branched at C3 or C4, amino acid substitutions at positions Leu239, Phe236 and Thr245 were introduced and resulted in mutant enzymes with completely different substrate specificities. The substitution T245A resulted in a relative shift of substrate specificity for keto-tert-leucine of more than 17000 compared with the 2-oxocaproate (kcat/KM). For the substrates branched at C4 a relative shift of up to 500 was obtained for several enzyme variants. A total of nine mutations were introduced and the kinetic data for the set of six substrates were determined for each of the resulting mutant enzymes. These were compared with those of the wild-type enzyme and rationalized by the active site model of L-HicDH. An analysis of the enzyme variants provided new insight into the residues involved in substrate binding and residues of importance for the differences between LDHs and HicDH. After the protein design project was complete the X-ray structure of the enzyme was solved in our group. A comparison between the model and the experimental 3D structure proved the quality of the model. All the variants were designed, expressed and tested before the 3D structure became available.