Tracking Down a New Steroid‐Hydroxylating Promiscuous Cytochrome P450: CYP154C8 from Streptomyces sp. W2233‐SM

CYP154C8 from Streptomyces sp. has been identified as a new cytochrome P450 with substrate flexibility towards different sets of steroids. In vitro treatment of these steroids with CYP154C8 revealed interesting product formation patterns with the same group of steroids. NMR study revealed the major product of corticosterone to be hydroxylated at the C21 position, whereas progesterone, androstenedione, testosterone, and 11‐ketoprogesterone were exclusively hydroxylated at the 16α position. However, the 16α‐hydroxylated product of progesterone was further hydroxylated to yield dihydroxylated products. 16‐hydroxyprogesterone was hydroxylated at two positions to yield dihydroxylated products: 2α,16α‐dihydroxyprogesterone and 6β,16α‐dihydroxyprogesterone. To the best of our knowledge, this is the first report of generation of such products through enzymatic hydroxylation by a CYP450. In view of the importance of modified steroids as pharmaceutical components, CYP154C8 has immense potential for utilization in bioproduction of hydroxylated derivative compounds to be directly employed for pharmaceutical applications.     

Theoretical and experimental evaluation of a CYP106A2 low homology model and production of mutants with changed activity and selectivity of hydroxylation

Steroids are important pharmaceutically active compounds. In contrast to the liver drug-metabolising cytochrome P450s, which metabolise a variety of substrates, steroid hydroxylases generally display a rather narrow substrate specificity. It is therefore a challenging goal to change their regio- and stereoselectivity. CYP106A2 is one of only a few bacterial steroid hydroxylases and hydroxylates 3-oxo-Delta4-steroids mainly in 15beta-position. In order to gain insights into the structure and function of this enzyme, whose crystal structure is unknown, a homology model has been created. The substrate progesterone was then docked into the active site to predict which residues might affect substrate binding. The model was substantiated by using a combination of theoretical and experimental investigations. First, numerous computational structure evaluation tools assessed the plausibility of its protein geometry and its quality. Second, the model explains many key properties of common cytochrome P450s. Third, two sets of mutants have been heterologously expressed, and the influence of the mutations on the catalytic activity towards deoxycorticosterone and progesterone has been studied experimentally: the first set comprises six mutations located in the structurally variable regions of this enzyme that are very difficult to predict by cytochrome P450 modelling (K27R, I86T, E90V, I71T, D185G and I215T). For these positions, no participation in the active-site formation was predicted, or could be experimentally demonstrated. The second set comprises five mutants in substrate recognition site 6 (S394I, A395L, T396R, G397P and Q398S). For these residues, participation in active-site formation and an influence on substrate binding was predicted by docking. These mutants are based on an alignment with human CYP11B1, and in fact most of these mutants altered the active-site structure and the hydroxylation activity of CYP106A2 dramatically.     

CYP154C5 Regioselectivity in Steroid Hydroxylation Explored by Substrate Modifications and Protein Engineering**

CYP154C5 from Nocardia farcinica is a P450 monooxygenase able to hydroxylate a range of steroids with high regio- and stereoselectivity at the 16α-position. Using protein engineering and substrate modifications based on the crystal structure of CYP154C5, an altered regioselectivity of the enzyme in steroid hydroxylation had been achieved. Thus, conversion of progesterone by mutant CYP154C5 F92A resulted in formation of the corresponding 21-hydroxylated product 11-deoxycorticosterone in addition to 16α-hydroxylation. Using MD simulation, this altered regioselectivity appeared to result from an alternative binding mode of the steroid in the active site of mutant F92A. MD simulation further suggested that the entrance of water to the active site caused higher uncoupling in this mutant. Moreover, exclusive 15α-hydroxylation was observed for wild-type CYP154C5 in the conversion of 5α-androstan-3-one, lacking an oxy-functional group at C17. Overall, our data give valuable insight into the structure–function relationship of this cytochrome P450 monooxygenase for steroid hydroxylation.     

Cutting Long Syntheses Short: Access to Non‐Natural Tyrosine Derivatives Employing an Engineered Tyrosine Phenol Lyase

The chemical synthesis of 3‐substituted tyrosine derivatives requires a minimum of four steps to access optically enriched material starting from commercial precursors. Attempting to short‐cut the cumbersome chemical synthesis of 3‐substituted tyrosine derivatives, a single step biocatalytic approach was identified employing the tyrosine phenol lyase from Citrobacter freundii. The enzyme catalyses the hydrolysis of tyrosine to phenol, pyruvate and ammonium as well as the reverse reaction, thus the formation of tyrosine from phenol, pyruvate and ammonium. Since the wild‐type enzyme possessed a very narrow substrate spectrum, structure‐guided, site‐directed mutagenesis was required to change the substrate specificity of this C C bond forming enzyme. The best variant M379V transformed, for example, o‐cresol, o‐methoxyphenol and o‐chlorophenol efficiently to the corresponding tyrosine derivatives without any detectable side‐product. In contrast, all three phenol compounds were non‐substrates for the wild‐type enzyme. Employing the mutant, various L ‐tyrosine derivatives (3‐Me, 3‐OMe, 3‐F, 3‐Cl) were obtained with complete conversion and excellent enantiomeric excess (>97%) in just a single ‘green’ step starting from pyruvate and commercially available phenol derivatives.     

An efficient route to selective bio-oxidation catalysts: an iterative approach comprising modeling, diversification, and screening, based on CYP102A1

Perillyl alcohol is the terminal hydroxylation product of the cheap and readily available terpene, limonene. It has high potential as an anti‐tumor substance, but is of limited availability. In principle, cytochrome P450 monooxygenases, such as the self‐sufficient CYP102A1, are promising catalysts for the oxidation of limonene or other inert hydrocarbons. The wild‐type enzyme converts (4R)‐limonene to four different oxidation products; however, terminal hydroxylation at the allylic C7 is not observed. Here we describe a generic strategy to engineer this widely used enzyme to hydroxylate exclusively the exposed, but chemically less reactive, primary C7 in the presence of other reactive positions. The approach presented here turns CYP102A1 into a highly selective catalyst with a shifted product spectra by successive rounds of modeling, the design of small focused libraries, and screening. In the first round a minimal CYP102A1 mutant library was rationally designed. It contained variants with improved or strongly shifted regio‐, stereo‐ and chemoselectivity, compared to wild‐type. From this library the variant with the highest perillyl alcohol ratio was fine‐tuned by two additional rounds of molecular modeling, diversification, and screening. In total only 29 variants needed to be screened to identify the triple mutant A264V/A238V/L437F that converts (4R)‐limonene to perillyl alcohol with a selectivity of 97 %. Focusing mutagenesis on a small number of relevant positions identified by computational approaches is the key for efficient screening for enzyme selectivity.     

Towards Preparative Scale Steroid Hydroxylation with Cytochrome P450 Monooxygenase CYP106A2

Cytochrome P450 monooxygenases are of outstanding interest for the synthesis of pharmaceuticals and fine chemicals, due to their ability to hydroxylate C? H bonds mainly in a stereo‐ and regioselective manner. CYP106A2 from Bacillus megaterium ATCC 13368, one of only a few known bacterial steroid hydroxylases, enables the oxidation of 3‐keto‐4‐ene steroids mainly at position 15. We expressed this enzyme together with the electron‐transfer partners bovine adrenodoxin and adrenodoxin reductase in Escherichia coli. Additionally an enzyme‐coupled cofactor regeneration system was implemented by expressing alcohol dehydrogenase from Lactobacillus brevis. By studying the conversion of progesterone and testosterone, the bottlenecks of these P450‐catalyzed hydroxylations were identified. Substrate transport into the cell and substrate solubility turned out to be crucial for the overall performance. Based on these investigations we developed a new concept for CYP106A2‐catalyzed steroid hydroxylations by which the productivity of progesterone and testosterone conversion could be increased up to 18‐fold to yield an absolute productivity up to 5.5 g L ^?1 d^?1. Product extraction with absorber resins allowed the recovery of quantitative amounts of 15β‐OH‐progesterone and 15β‐OH‐testosterone and also the reuse of the biocatalyst.     

Structural basis for the properties of two single-site proline mutants of CYP102A1 (P450BM3)

The crystal structures of the haem domains of Ala330Pro and Ile401Pro, two single‐site proline variants of CYP102A1 (P450_BM3) from Bacillus megaterium, have been solved. In the A330P structure, the active site is constricted by the relocation of the Pro329 side chain into the substrate access channel, providing a basis for the distinctive C? H bond oxidation profiles given by the variant and the enhanced activity with small molecules. I401P, which is exceptionally active towards non‐natural substrates, displays a number of structural similarities to substrate‐bound forms of the wild‐type enzyme, notably an off‐axial water ligand, a drop in the proximal loop, and the positioning of two I‐helix residues, Gly265 and His266, the reorientation of which prevents the formation of several intrahelical hydrogen bonds. Second‐generation I401P variants gave high in vitro oxidation rates with non‐natural substrates as varied as fluorene and propane, towards which the wild‐type enzyme is essentially inactive. The substrate‐free I401P haem domain had a reduction potential slightly more oxidising than the palmitate‐bound wild‐type haem domain, and a first electron transfer rate that was about 10 % faster. The electronic properties of A330P were, by contrast, similar to those of the substrate‐free wild‐type enzyme.     

Controlling the Regioselectivity of Baeyer–Villiger Monooxygenases by Mutation of Active‐Site Residues

Baeyer–Villiger monooxygenase (BVMO)‐mediated regiodivergent conversions of asymmetric ketones can lead to the formation of “normal” or “abnormal” lactones. In a previous study, we were able to change the regioselectivity of a BVMO by mutation of the active‐site residues to smaller amino acids, which thus created more space. In this study, we demonstrate that this method can also be used for other BVMO/substrate combinations. We investigated the regioselectivity of 2‐oxo‐Δ³‐4,5,5‐trimethylcyclopentenylacetyl‐CoA monooxygenase from Pseudomonas putida (OTEMO) for cis‐bicyclo[3.2.0]hept‐2‐en‐6‐one ( 1 ) and trans‐dihydrocarvone ( 2 ), and we were able to switch the regioselectivity of this enzyme for one of the substrate enantiomers. The OTEMO wild‐type enzyme converted (?)‐ 1 into an equal (50:50) mixture of the normal and abnormal products. The F255A/F443V variant produced 90 % of the normal product, whereas the W501V variant formed up to 98 % of the abnormal product. OTEMO F255A exclusively produced the normal lactone from (+)‐ 2 , whereas the wild‐type enzyme was selective for the production of the abnormal product. The positions of these amino acids were equivalent to those mutated in the cyclohexanone monooxygenases from Arthrobacter sp. and Acinetobacter sp. (CHMO_Arthro and CHMO_Acineto) to switch their regioselectivity towards (+)‐ 2 , which suggests that there are hot spots in the active site of BVMOs that can be targeted with the aim to change the regioselectivity.     

Control of Lipase Enantioselectivity by Engineering the Substrate Binding Site and Access Channel

Lipase from Burkholderia cepacia (BCL) has proven to be a very useful biocatalyst for the resolution of 2‐substituted racemic acid derivatives, which are important chiral building blocks. Our previous work showed that enantioselectivity of the wild‐type BCL could be improved by chemical engineering of the substrate's molecular structure. From this earlier study, three amino acids (L17, V266 and L287) were proposed as targets for mutagenesis aimed at tailoring enzyme enantioselectivity. In the present work, a small library of 57 BCL single mutants targeted on these three residues was constructed and screened for enantioselectivity towards (R,S)‐2‐chloro ethyl 2‐bromophenylacetate. This led to the fast isolation of three single mutants with a remarkable tenfold enhanced or reversed enantioselectivity. Analysis of substrate docking and access trajectories in the active site was then performed. From this analysis, the construction of 13 double mutants was proposed. Among them, an outstanding improved mutant of BCL was isolated that showed an E value of 178 and a 15‐fold enhanced specific activity compared to the parental enzyme; thus, this study demonstrates the efficiency of the semirational engineering strategy.     

Engineering the CYP101 system for in vivo oxidation of unnatural substrates

The protein engineering of CYP enzymes for structure–activitystudies and the oxidation of unnatural substrates for biotechnologicalapplications will be greatly facilitated by the availabilityof functional, whole-cell systems for substrate oxidation. Wereport the construction of a tricistronic plasmid that expressesthe CYP101 monooxygenase from Pseudomonas putida, and its physiologicalelectron transfer co-factor proteins putidaredoxin reductaseand putidaredoxin in Escherichia coli, giving a functional invivo catalytic system. Wild-type CYP101 expressed in this systemefficiently transforms camphor to 5-exo-hydroxycamphor withoutfurther oxidation to 5-oxo-camphor until >95% of camphorhas been consumed. CYP101 mutants with increased activity forthe oxidation of diphenylmethane (the Y96F–I395G mutant),styrene and ethylbenzene (the Y96F–V247L mutant) havebeen engineered. In particular, the Y96F–V247L mutantshows coupling efficiency of approximately 60% for styrene andethylbenzene oxidation, with substrate oxidation rates of approximately100/min. Escherichia coli cells transformed with tricistronicplasmids expressing these mutants readily gave 100-mg quantitiesof 4-hydroxydiphenylmethane and 1-phenylethanol in 24–72h. This new in vivo system can be used for preparative scalereactions for product characterization, and will greatly facilitatedirected evolution of the CYP101 enzyme for enhanced activityand selectivity of substrate oxidation.     

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 Oxidation of Hydrophobic Aromatic Substrates by Using a Variant of the P450 Monooxygenase CYP101B1

The cytochrome P450 monooxygenase CYP101B1, from a Novosphingobium bacterium is able to bind and oxidise aromatic substrates but at a lower activity and efficiency than norisoprenoids and monoterpenoid esters. Histidine 85 of CYP101B1 aligns with tyrosine 96 of CYP101A1, which, in the latter enzyme forms the only hydrophilic interaction with its substrate, camphor. The histidine residue of CYP101B1 was mutated to phenylalanine with the aim of improving the activity of the enzyme for hydrophobic substrates. The H85F mutant lowered the binding affinity and activity of the enzyme for β-ionone and altered the oxidation selectivity. This variant also showed enhanced affinity and activity towards alkylbenzenes, styrenes and methylnaphthalenes. For example the rate of product formation for acenaphthene oxidation was improved sixfold to 245 nmol per nmol CYP per min. Certain disubstituted naphthalenes and substrates, such as phenylcyclohexane and biphenyls, were oxidised with lower activity by the H85F variant. Variants at H85 (A and G) designed to introduce additional space into the active site so as to accommodate these larger substrates did not improve the oxidation activity. As the H85F mutant of CYP101B1 improved the oxidation of hydrophobic substrates, this residue is likely to be in the substrate binding pocket or the access channel of the enzyme. The side chain of the histidine might interact with the carbonyl groups of the favoured norisoprenoid substrates of CYP101B1.     

Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone

CYP106A2 is known as a 15β‐hydroxylase, but also shows minor 11α‐hydroxylase activity for progesterone. 11α‐Hydroxyprogesterone is an important pharmaceutical compound with anti‐androgenic and blood‐pressure‐regulating activity. This work therefore focused on directing the regioselectivity of the enzyme towards hydroxylation at position 11 in the C ring of the steroid through a combination of saturation mutagenesis and rational site‐directed mutagenesis. With the aid of data from a homology model of CYP106A2 containing docked progesterone, together with site‐directed mutagenesis of active‐site residues (Lisurek et al. ChemBioChem 2008 , 9, 1439–1449), a saturation mutagenesis library at positions A395 and G397 was created. Screening of the library identified the mutants A395I and A395W/G397K as having 11α‐hydroxylase activities 8.9 and 11.5 times higher than that of the wild type (WT). In the next step, additional mutations were integrated by a rational site‐directed mutagenesis approach to increase the catalytic efficiency. Of the 40 candidates analyzed, the mutants A106T/A395I, A106T/A395I/R409L, and T89N/A395I turned out to display increased 11α‐hydroxylase selectivities and activities relative to the WT (14.3‐, 12.6‐, and 11.8‐fold increases in selectivity and 39.3‐, 108‐, and 24.4‐ in k_cat/K_m). In the last step of the study, the best mutants were applied in a whole‐cell biotransformation. In these experiments the production (percentage) of 15β‐hydroxyprogesterone decreased from 50.4 % (wild type) to 4.8 % (mutant T89N/A395I), whereas that of 11α‐hydroxyprogesterone increased from 27.7 to 80.9 %, thus demonstrating an impressive regioselectivity.     

杜仲中环烯醚萜类物质对性激素转化的调控作用

杜仲是中国传统名贵滋补药材,有补益肝肾、强筋壮骨、固经安胎的功效,而这些功效与性激素水平息息相关,故本文研究了杜仲中3种主要的环烯醚萜类物质（京尼平、京尼平苷和京尼平苷酸）对性激素的转化作用。以卵巢颗粒细胞的肿瘤细胞系KGN作为激素的转化系统,孕烯醇酮为转化底物,分别测量环烯醚萜类物质干预24h后培养基中的孕酮、睾酮以及雌二醇的浓度,并提取细胞的RNA,以反转录实时荧光定量法来研究其催化酶3β-HSD、CYP17A1以及17β-HSD表达水平的影响。研究结果表明,50 μmol/L的京尼平能显著提升3β-HSD、CYP17A1以及17β-HSD的表达水平,从而显著促进睾酮和雌二醇的合成。     

Functional Investigations of Thromboxane Synthase (CYP5A1) in Lipid Bilayers of Nanodiscs

CYP5A1 is a membrane‐associated cytochrome P450 that metabolizes the cyclooxygenase product prostaglandin (PGH₂) into thromboxane A₂ (TXA₂), a potent inducer of vasoconstriction and platelet aggregation. Although CYP5A1 is an ER‐bound protein, the role of membranes in modulating the thermodynamics and kinetics of substrate binding to this protein has not been elucidated. In this work, we incorporated thromboxane synthase into lipid bilayers of nanodiscs for functional studies. We measured the redox potential of CYP5A1 in nanodiscs and showed that the redox potential is within a similar range of other drug‐metabolizing P450 enzymes in membranes. Further, we showed that binding of substrate to CYP5A1 can induce conformational changes in the protein that block small‐molecule ligand egress by measuring the kinetics of cyanide binding to CYP5A1 as a function of substrate concentration. Notably, we observed that sensitivity to cyanide binding was different for two substrate analogues, U44069 and U46619, thus indicating that they bind differently to the active site of CYP5A1. We also characterized the effects of the different lipids on CYP5A1 catalytic activity by using nanodiscs to create unary, binary, and ternary lipid systems. CYP5A1 activity increased dramatically in the presence of charged lipids POPS and POPE, as compared to the unary POPC system. These results suggest the importance of lipid composition on modulating the activity of CYP5A1 to increase thromboxane formation.     

Immobilization of α‐chymotrypsin for use in batch and continuous reactors

α‐Chymotrypsin from bovine pancrease (EC 3.4.21.1) was entrapped in Ca‐alginate gel particles to carry out hydrolysis of N‐acetyl‐L ‐phenylalanine methyl ester (APME) in batch as well as continuous fixed bed reactor. The enzyme was covalently modified with homo‐bifunctional polyethylene glycol derivatives in order to reduce its leakage from the beads; 85% modification of the ∈‐NH₂ groups of lysine residues caused reduction in the enzyme activity by 50%. However, this modification was helpful in a long run because it reduced both enzyme leakage and deactivation. Effective diffusivities and the distribution coefficients of the substrate and the product were determined experimentally, and later used in simulation of a batch experiment employing the beads. A continuous fixed bed reactor with the gel beads was operated to study the deactivation of the enzyme. During a 15‐day period, the enzyme showed about 15% loss in the conversion which occurred only during the first 5 days. After that the enzyme did not deactivate further which demonstrates that this method can be applied for continuous reactions. © 2000 Society of Chemical Industry     

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.      

Substrate Binding Drives Active‐Site Closing of Human Blood Group B Galactosyltransferase as Revealed by Hot‐Spot Labeling and NMR Spectroscopy Experiments

Crystallography has shown that human blood group A (GTA) and B (GTB) glycosyltransferases undergo transitions between “open”, “semiclosed”, and “closed” conformations upon substrate binding. However, the timescales of the corresponding conformational reorientations are unknown. Crystal structures show that the Trp and Met residues are located at “conformational hot spots” of the enzymes. Therefore, we utilized ¹⁵N side‐chain labeling of Trp residues and ¹³C‐methyl labeling of Met residues to study substrate‐induced conformational transitions of GTB. Chemical‐shift perturbations (CSPs) of Met and Trp residues in direct contact with substrate ligands reflect binding kinetics, whereas the CSPs of Met and Trp residues at remote sites reflect conformational changes of the enzyme upon substrate binding. Acceptor binding is fast on the chemical‐shift timescale with rather small CSPs in the range of less than approximately 20 Hz. Donor binding matches the intermediate exchange regime to yield an estimate for exchange rate constants of approximately 200–300 Hz. Donor or acceptor binding to GTB saturated with acceptor or donor substrate, respectively, is slow (<10 Hz), as are coupled protein motions, reflecting mutual allosteric control of donor and acceptor binding. Remote CSPs suggest that substrate binding drives the enzyme into the closed state required for catalysis. These findings should contribute to better understanding of the mechanism of glycosyl transfer of GTA and GTB.     

Enzymatic Hydrolysis of Corn Stover after Pretreatment with Dilute Sulfuric Acid

Although many previous studies have been carried on the enzymatic hydrolysis of corn stover after pretreatment with dilute sulfuric acid, this paper emphasizes the use of different conditions to attain the highest yields of two sugars, xylose and glucose, from both stages. The pretreatment was performed at a range of sulfuric acid concentrations of 2, 4 and 6 % at 80, 100 and 120 °C. Up to 77 % xylose yield was obtained while the glucose yield was only 8.4 %. The corresponding solid phase was hydrolyzed by cellulase and the influences of five factors and their interactions on enzyme hydrolysis were evaluated by response surface methodology based on one‐factor‐at‐a‐time experiments. The optimal levels for each variable to obtain the highest reducing sugar yield were as follows: enzyme concentration of 22 FPU/g substrate, substrate concentration of 77 g/L, temperature of 49 °C, pH 4.8 and reaction time of 38 h. A reducing sugar yield of 42.11 g/100 g substrate was achieved, which was consistent with the predicted value of 42.13 g/100 g substrate. Compared with the one‐factor‐at‐a‐time experiments, there was a 9.4 % increase in reducing sugar yield when the enzyme concentration was decreased to 3 FPU/g substrate, the substrate concentration increased to 17 g/L and the reaction time dropped to 22 h. The total sugar released from the two stages was 62.81 g/100 g substrate.     

Revisiting Cytochrome P450‐Mediated Oxyfunctionalization of Linear and Cyclic Alkanes

Cytochrome P450 monooxygenases (CYPs) of the CYP153 family catalyse terminal hydroxylation of n‐alkanes. Alkane hydroxylating mutants of self‐sufficient CYP102A1 have also been described. We evaluated two CYP153s (a three‐component system and a fused self‐sufficient CYP), wild‐type CYP102A1 and nine CYP102A1 mutants, for the conversion of three cycloalkanes (C₆, C₇ and C₈) and three n‐alkanes (C₆, C₈ and C₁₀) using whole cells (WCs) and crude cell‐free extracts (CFEs). The aim was to identify substrate–enzyme combinations that give high product titres and space‐time yields (STYs). Comparisons were made using total turnover numbers (TTNs) and turnover frequencies (TOFs) to normalize for CYP expression. Reactions were carried out using high enzyme and substrate concentrations compatible with high STYs. Under these conditions CYP102A1 and the double R47L,Y51F mutant, although not regioselective, performed better on all substrates in terms of product titres over 8 h, and thus STYs and TTNs, than heavily mutated variants that have been reported to give very high TOFs. CYP153A6, with its ferredoxin (Fdx) and ferredoxin reductase (FdR), emerged as the superior catalyst for conversion of n‐alkanes. In addition to its excellent regioselectivity it also gave the highest final product titres and STYs in WC conversions of hexane and octane. Interaction with FdR and Fdx initially limited performance in CFEs, but with additional FdR and Fdx gave 1‐octanol titres of 50 mmol⋅L_BRM⁻¹ and TTNs exceeding 12,000 over 18 h, rivalling results reported with self‐sufficient CYPs. Selecting biocatalysts for application requires caution, since experimental conditions such as amount of substrate added and solubility as well as cofactor dependence and regeneration can have a profound effect on catalyst performance, while stability and efficiency with regard to cofactor usage (coupling efficiency) are at least as important as TOFs when high product titres and STYs are the target.