Structural and Mutational Studies on the Unusual Substrate Specificity of meso‐Diaminopimelate Dehydrogenase from Symbiobacterium thermophilum

Wild‐type meso‐diaminopimelate dehydrogenase (DAPDH) is usually specific to the native substrate, meso‐2,6‐diaminopimelate. Recently, a DAPDH from Symbiobacterium thermophilum (StDAPDH) was found to exhibit expanded substrate specificity. As such, its crystal structures in apo form and in complex with NADP⁺ and both NADPH and meso‐DAP were investigated to reveal the structural basis of its unique catalytic properties. Structural analysis results show that StDAPDH should prefer an ordered kinetic catalytic mechanism. A second substrate entrance tunnel with Met152 at its bottleneck was found, through which pyruvate/D ‐alanine might bind and enter the catalytic cavity, providing some structural insights into its high activity toward pyruvate. The side chain of Met152 might interact with Asp92 and Asn253, thus affecting the domain motion and catalysis. These results offer useful information for understanding the unique catalytic properties of StDAPDH and guiding further engineering of this enzyme.     

Investigation of Structural Determinants for the Substrate Specificity in the Zinc‐Dependent Alcohol Dehydrogenase CPCR2 from Candida parapsilosis

Zinc‐dependent alcohol dehydrogenases (ADHs) are a class of enzymes applied in different biocatalytic processes ranging from lab to industrial scale. However, one drawback is the limited substrate range, necessitating a whole array of different ADHs for the relevant substrate classes. In this study, we investigated structural determinants of the substrate spectrum in the zinc‐dependent ADH carbonyl reductase 2 from Candida parapsilosis (CPCR2), combining methods of mutational analysis with in silico substrate docking. Assigned active site residues were genetically randomized, and the resulting mutant libraries were screened with a selection of challenging carbonyl substrates. Three variants (C57A, W116K, and L119M) with improved activities toward different substrates were detected at neighboring positions in the active site. Thus, all possible combinations of the mutations were generated and characterized for their substrate specificity, yielding several improved variants. The most interesting were a C57A variant, with a 27‐fold increase in specific activity for 4′‐acetamidoacetophenone, and the double mutant CPCR2 B16‐(C57A, L119M), with a 45‐fold improvement in the k_cat?K_M^?1 value. The obtained variants were further investigated by in silico docking experiments. The results indicate that the mentioned residues are structural determinants of the substrate specificity of CPCR2, being major players in the definition of the active site. Comparison of these results with closely related enzymes suggests that these might even be transferred to other ADHs.     

Rational Engineering of Hydratase from Lactobacillus acidophilus Reveals Critical Residues Directing Substrate Specificity and Regioselectivity

Enzymatic conversion of fatty acids (FAs) by fatty acid hydratases (FAHs) presents a green and efficient route for high-value hydroxy fatty acid (HFA) production. However, limited diversity was achieved among HFAs, to date, with respect to chain length and hydroxy position. In this study, two highly similar FAHs from Lactobacillus acidophilus were compared: FA-HY2 has a narrow substrate scope and strict regioselectivity, whereas FA-HY1 utilizes longer chain substrates and hydrates various double-bond positions. It is revealed that three active-site residues play a remarkable role in directing substrate specificity and regioselectivity of hydration. If these residues on FA-HY2 are mutated to the corresponding ones in FA-HY1, a significant expansion of substrate scope and a distinct enhancement in hydration of double bonds towards the ω-end of FAs is observed. A three-residue mutant of FA-HY2 (TM-FA-HY2) displayed an impressive reversal of regioselectivity towards linoleic acid, shifting the ratio of the HFA regioisomers (10-OH/13-OH) from 99:1 to 12:88. Notable changes in regioselectivity were also observed for arachidonic acid and for C₁₈ polyunsaturated fatty acid substrates. In addition, TM-FA-HY2 converted eicosapentaenoic acid into its 12-hydroxy product with high conversion at the preparative scale. Furthermore, it is demonstrated that microalgae are a source of diverse FAs for HFA production. This study paves the way for tailor-made FAH design to enable the production of diverse HFAs for various applications from the polymer industry to medical fields.     

Expanding Substrate Specificity of ω‐Transaminase by Rational Remodeling of a Large Substrate‐Binding Pocket

Production of structurally diverse chiral amines via biocatalytic transamination is challenged by severe steric interference in a small active site pocket of ω‐transaminase (ω‐TA). Herein, we demonstrated that structure‐guided remodeling of a large pocket by a single point mutation, instead of excavating the small pocket, afforded desirable alleviation of the steric constraint without deteriorating parental activities toward native substrates. Molecular modeling suggested that the L57 residue of the ω‐TA from Ochrobactrum anthropi acted as a latch that forced bulky substrates to undergo steric interference with the small pocket. Removal of the latch by a L57A substitution allowed relocation of the small pocket and dramatically improved activities toward various arylalkylamines and alkylamines (e.g., 1100‐fold increase in k_cat/K_M for α‐propylbenzylamine). This approach may provide a facile strategy to broaden the substrate specificity of ω‐TAs.

     

Structural Determinants for the Non‐Canonical Substrate Specificity of the ω‐Transaminase from Paracoccus denitrificans

Substrate binding pockets of ω‐transaminase (ω‐TA) consist of a large (L) pocket capable of dual recognition of hydrophobic and carboxyl substituents, and a small (S) pocket displaying a strict steric constraint that permits entry of a substituent no larger than an ethyl group. Despite the unique catalytic utility of ω‐TA enabling asymmetric reductive amination of carbonyl compounds, the severe size exclusion occurring in the S pocket has limited synthetic applications of ω‐TA to access structurally diverse chiral amines and amino acids. Here we report the first example of an ω‐TA whose S pocket shows a non‐canonical steric constraint and readily accommodates up to an n‐butyl substituent. The relaxed substrate specificity of the (S)‐selective ω‐TA, cloned from Paracoccus denitrificans (PDTA), afforded efficient asymmetric syntheses of unnatural amino acids carrying long alkyl side chains such as L ‐norvaline and L ‐norleucine. Molecular modeling using the recently released X‐ray structure of PDTA could pinpoint an exact location of the S pocket which had remained dubious. Entry of a hydrophobic substituent in the L pocket was found to have the S pocket accept up to an ethyl substituent, reminiscent of the canonical steric constraint. In contrast, binding of a carboxyl group to the L pocket induced a slight movement of V153 away from the small‐pocket‐forming residues. The resulting structural change elicited excavation of the S pocket, leading to formation of a narrow tunnel‐like structure allowing accommodation of linear alkyl groups of carboxylate‐bearing substrates. To verify the active site model, we introduced site‐directed mutagenesis to six active site residues and examined whether the point mutations alleviated the steric constraint in the S pocket. Consistent with the molecular modeling results, the V153A variant assumed an elongated S pocket and accepted even an n‐hexyl substituent. Our findings provide precise structural information on substrate binding to the active site of ω‐TA, which is expected to benefit rational redesign of substrate specificity of ω‐TA.

     

Kinetic Analysis of PRMT1 Reveals Multifactorial Processivity and a Sequential Ordered Mechanism

Arginine methylation is a prevalent post‐translational modification in eukaryotic cells. Two significant debates exist within the field: do these enzymes dimethylate their substrates in a processive or distributive manner, and do these enzymes operate using a random or sequential method of bisubstrate binding? We revealed that human protein arginine N‐methyltransferase 1 (PRMT1) enzyme kinetics are dependent on substrate sequence. Further, peptides containing an Nη‐hydroxyarginine generally demonstrated substrate inhibition and had improved K_M values, which evoked a possible role in inhibitor design. We also revealed that the perceived degree of enzyme processivity is a function of both cofactor and enzyme concentration, suggesting that previous conclusions about PRMT sequential methyl transfer mechanisms require reassessment. Finally, we demonstrated a sequential ordered Bi–Bi kinetic mechanism for PRMT1, based on steady‐state kinetic analysis. Together, our data indicate a PRMT1 mechanism of action and processivity that might also extend to other functionally and structurally conserved PRMTs.     

Catalytic Mechanism of the Hotdog‐Fold Thioesterase PA1618 Revealed by X‐ray Structure Determination of a Substrate‐Bound Oxygen Ester Analogue Complex

Thioesterase activity accounts for the majority of the activities in the hotdog‐fold superfamily. The structures and mechanisms of catalysis for many hotdog enzymes have been elucidated by X‐ray crystallography and kinetics to probe the specific substrate usage and cellular functions. However, structures of hotdog thioesterases in complexes with substrate analogues reported to date utilize ligands that either represent truncations of the substrate or include additional atoms to prevent hydrolysis. Here we present the synthesis of an isosteric and isoelectronic substrate analogue—benzoyl‐OdCoA—and the X‐ray crystal structure of a complex of the analogue with Pseudomonas aeruginosa hotdog thioesterase PA1618 (at 1.72 Å resolution). The complex is compared with that of the “imperfect” substrate analogue phenacyl‐CoA, refined to a resolution of 1.62 Å. Kinetic and structural results are consistent with Glu64 as the catalytic residue and with the involvement of Gln49 in stabilization of the transition state. Structural comparison of the two ligand‐bound structures revealed a crucial ordered water molecule coordinated in the active site of the benzoyl‐OdCoA structure but not present in the phenacyl‐CoA‐bound structure. This suggests a general base mechanism of catalysis in which Glu64 activates the coordinated water nucleophile. Together, our findings reveal the importance of a closely similar substrate analogue to determine the true substrate binding and catalytic mechanism.     

Modification of Substrate Specificity Resulting in an Epoxide Hydrolase with Shifted Enantiopreference for (2,3‐Epoxypropyl)benzene

Random mutagenesis targeted at hotspots of noncatalytic active‐site residues of potato epoxide hydrolase StEH1 combined with an enzyme‐activity screen allowed the isolation of enzyme variants displaying altered enantiopreference in the catalyzed hydrolysis of (2,3‐epoxypropyl)benzene. The wild‐type enzyme favored the S enantiomer with a ratio of 2.5:1, whereas the variant displaying the most radical functional changes showed a 15:1 preference for the R enantiomer. This mutant had accumulated four substitutions distributed over two out of four mutated hotspots: W106L, L109Y, V141K, and I151V. The underlying causes of the enantioselectivity were a decreased catalytic efficiency in the catalyzed hydrolysis of the S enantiomer combined with retained activity with the R enantiomer. The results demonstrate the feasibility of molding the stereoselectivity of this biocatalytically relevant enzyme.     

Mutant Lipase‐Catalyzed Kinetic Resolution of Bulky Phenyl Alkyl sec‐Alcohols: A Thermodynamic Analysis of Enantioselectivity

The size of the stereoselectivity pocket of Candida antarctica lipase B limits the range of alcohols that can be resolved with this enzyme. These steric constrains have been changed by increasing the size of the pocket by the mutation W104A. The mutated enzyme has good activity and enantioselectivity toward bulky secondary alcohols, such as 1‐phenylalkanols, with alkyl chains up to eight carbon atoms. The S enantiomer was preferred in contrast to the wild‐type enzyme, which has R selectivity. The magnitude of the enantioselectivity changes in an interesting way with the chain length of the alkyl moiety. It is governed by interplay between entropic and enthalpic contributions and substrates with long alkyl chains are resolved best with E values higher than 100. The enantioselectivity increases with temperature for the small substrates, but decreases for the long ones.     

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.     

The Structure of LiuC,a 3‐Hydroxy‐3‐Methylglutaconyl CoA Dehydratase Involved in Isovaleryl‐CoA Biosynthesis in Myxococcus xanthus,Reveals Insights into Specificity and Catalysis

Myxobacteria are able to produce the important metabolite isovaleryl coenzyme A by a route other than leucine degradation. The first step into this pathway is mediated by LiuC, a member of the 3‐methylglutaconyl CoA hydratases (MGCH). Here we present crystal structures refined to 2.05 and 1.1 Å of LiuC in the apo form and bound to coenzyme A, respectively. By using simulated annealing we modeled the enzyme substrate complex and identified residues potentially involved in substrate binding, specificity, and catalysis. The dehydration of 3‐hydroxy‐3‐methylglutaconyl CoA to 3‐methylglutaconyl CoA catalyzed by LiuC involves Glu112 and Glu132 and likely employs the typical crotonase acid‐base mechanism. In this, Tyr231 and Arg69 are key players in positioning the substrate to enable catalysis. Surprisingly, LiuC shows higher sequence and structural similarity to human MGCH than to bacterial forms, although they convert the same substrate. This study provides structural insights into the alternative isovaleryl coenzyme A biosynthesis pathway and might open a path for biofuel research, as isovaleryl‐CoA is a source for isobutene, a precursor for renewable fuels and chemicals.     

Designed RNAs with Two Peptide‐Binding Units as Artificial Templates for Native Chemical Ligation of RNA‐Binding Peptides

The template effect plays important roles not only in modern synthetic and enzymatic catalysis but also in the ancient “RNA‐polypeptide (RNP) world,” which has been postulated to be a crucial stage in the origin of life. To mimic primitive template catalysis of peptide ligations by RNAs, we previously reported the design and synthesis of a ternary RNP complex in which the ligation of two peptides was significantly facilitated by a template RNA with two peptide‐binding units. However, RNA molecules also promoted the ligation reaction in a nonspecific manner through electrostatic interactions between RNA and basic peptides. In this study, we suppressed this effect by reducing the length of the original template derived from the Tetrahymena intron RNA. This modification, however, decreased the template ability for the specific reaction. As an alternative RNA that was as effective as the original template, we found that a self‐dimerizing RNA was a promising template for peptide ligation without a nonspecific effect.     

Identification and Characterization of a Methionine γ‐Lyase in the Calicheamicin Biosynthetic Cluster of Micromonospora echinospora

CalE6 is a previously uncharacterized protein involved in the biosynthesis of calicheamicins in Micromonospora echinospora. It is a pyridoxal‐5′‐phosphate‐dependent enzyme and exhibits high sequence homology to cystathionine γ‐lyases and cystathionine γ‐synthases. However, it was found to be active towards methionine and to convert this amino acid into α‐ketobutyrate, ammonium, and methanethiol. The crystal structure of the cofactor‐bound holoenzyme was resolved at 2.0 Å; it contains two active site residues, Gly105 and Val322, specific for methionine γ‐lyases. Modeling of methionine into the active site allows identification of the active site residues responsible for substrate recognition and catalysis. These findings support that CalE6 is a putative methionine γ‐lyase producing methanethiol as a building block in biosynthesis of calicheamicins.     

Driving Force Analysis of Proton Tunnelling Across a Reactivity Series for an Enzyme‐Substrate Complex

Quantitative structure‐activity relationships are widely used to probe C? H bond breakage by quinoprotein enzymes.^[1–4] However, we showed recently that p‐substituted benzylamines are poor reactivity probes for the quinoprotein aromatic amine dehydrogenase (AADH) because of a requirement for structural change in the enzyme‐substrate complex prior to C? H bond breakage.^[5] This rearrangement is partially rate limiting, which leads to deflated kinetic isotope effects for p‐substituted benzylamines. Here we report reactivity (driving force) studies of AADH with p‐substituted phenylethylamines for which the kinetic isotope effect (～16) accompanying C? H/C? ²H bond breakage is elevated above the semi‐classical limit. We show bond breakage occurs by quantum tunnelling and that within the context of the environmentally coupled framework for H‐tunnelling the presence of the p‐substituent places greater demand on the apparent need for fast promoting motions. The crystal structure of AADH soaked with phenylethylamine or methoxyphenylethylamine indicates that the structural change identified with p‐substituted benzylamines should not limit the reaction with p‐substituted phenylethylamines. This is consistent with the elevated kinetic isotope effects measured with p‐substituted phenylethylamines. We find a good correlation in the rate constant for proton transfer with bond dissociation energy for the reactive C? H bond, consistent with a rate that is limited by a Marcus‐like tunnelling mechanism. As the driving force becomes larger, the rate of proton transfer increases while the Marcus activation energy becomes smaller. This is the first experimental report of the driving force perturbation of H‐tunnelling in enzymes using a series of related substrates. Our study provides further support for proton tunnelling in AADH.     

Single‐Biocatalyst Synthesis of Enantiopure d‐Arylalanines Exploiting an Engineered d‐Amino Acid Dehydrogenase

A practical and efficient biocatalytic synthesis of aromatic d ‐amino acids has been developed, based on the reductive amination of the corresponding α‐keto acids via a recombinant whole cell system composed of an engineered dehydrogenase and cofactor recycling apparatus. The reaction was shown to give excellent enantioselectivity (≥98%) and good yields at the preparative scale across a broad range of substrates. Additionally, the structure of the variant enzyme was solved to allow rationalisation of the observed reaction rates. The engineered whole cell catalyst was also used to mediate the production of d ‐phenylalanine derivatives from racemic mixtures and cheaper l ‐amino acids by combining it with an enantiocomplementary deaminase.

     

Structural and Catalytic Characterization of Pichia stipitis OYE 2.6, a Useful Biocatalyst for Asymmetric Alkene Reductions

We have probed Pichia stipitis CBS 6054 Old Yellow Enzyme 2.6 (OYE 2.6) by several strategies including X‐ray crystallography, ligand binding and catalytic assays using the wild‐type as well as libraries of site‐saturation mutants. The alkene reductase crystallized in space group P 6₃ 2 2 with unit cell dimensions of 127.1×123.4 Å and its structure was solved to 1.5 Å resolution by molecular replacement. The protein environment surrounding the flavin mononucleotide (FMN) cofactor was very similar to those of other OYE superfamily members; however, differences in the putative substrate binding site were also observed. Substrate analog complexes were analyzed by both UV‐Vis titration and X‐ray crystallography to provide information on possible substrate binding interactions. In addition, four active site residues were targeted for site saturation mutagenesis (Thr 35, Ile 113, His 188, His 191) and each library was tested against three representative Baylis–Hillman adducts. Thr 35 could be replaced by Ser with no change in activity; other amino acids (Ala, Cys, Leu, Met, Gln and Val) resulted in diminished catalytic efficiency. The Ile 113 replacement library yielded a range of catalytic activities, but had very little impact on stereoselectivity. Finally, the two His residues (188 and 191) were essentially intolerant of substitutions with the exception of the His 191 Asn mutant, which did show significant catalytic ability. Structural comparisons between OYE 2.6 and Saccharomyces pastorianus OYE1 suggest that the key interactions between the substrate hydroxymethyl groups and the side‐chain of Thr 35 and/or Tyr 78 play an important role in making OYE 2.6 an (S)‐selective alkene reductase.