Gerrit J. Poelarends

Halohydrin Dehalogenases Are Structurally and Mechanistically Related to Short-Chain Dehydrogenases/Reductases

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.

The Lactococcal lmrP Gene Encodes a Proton Motive Force- dependent Drug Transporter*

To genetically dissect the drug extrusion systems of Lactococcus lactis, a chromosomal DNA library was made in Escherichia coli and recombinant strains were selected for resistance to high concentrations of ethidium bromide. Recombinant strains were found to be resistant not only to ethidium bromide but also to daunomycin and tetraphenylphosphonium. The drug resistance is conferred by the lmrP gene, which encodes a hydrophobic polypeptide of 408 amino acid residues with 12 putative membrane-spanning segments. Some sequence elements in this novel membrane protein share similarity to regions in the transposon Tn10-encoded tetracycline resistance determinant TetA, the multidrug transporter Bmr from Bacillus subtilis, and the bicyclomycin resistance determinant Bcr from E. coli. Drug resistance associated with lmrP expression correlated with energy-dependent extrusion of the molecules. Drug extrusion was inhibited by ionophores that dissipate the proton motive force but not by the ATPase inhibitor ortho-vanadate. These observations are indicative for a drug-proton antiport system. A lmrP deletion mutant was constructed via homologous recombination using DNA fragments of the flanking region of the gene. The L. lactis (ΔlmrP) strain exhibited residual ethidium extrusion activity, which in contrast to the parent strain was inhibited by ortho-vanadate. The results indicate that in the absence of the functional drug-proton antiporter LmrP, L. lactis is able to overexpress another, ATP-dependent, drug extrusion system. These findings substantiate earlier studies on the isolation and characterization of drug-resistant mutants of L. lactis (Bolhuis, H., Molenaar, D., Poelarends, G., van Veen, H. W., Poolman, B., Driessen, A. J. M., and Konings, W. N.(1994) J. Bacteriol. 176, 6957-6964).

Roles of horizontal gene transfer and gene integration in evolution of 1,3-dichloropropene- and 1,2-dibromoethane-degradative pathways.

The haloalkane-degrading bacteria Rhodococcus rhodochrous NCIMB13064, Pseudomonas pavonaceae 170, and Mycobacterium sp. strain GP1 share a highly conserved haloalkane dehalogenase gene (dhaA). Here, we describe the extent of the conserved dhaA segments in these three phylogenetically distinct bacteria and an analysis of their flanking sequences. The dhaA gene of the 1-chlorobutane-degrading strain NCIMB13064 was found to reside within a 1-chlorobutane catabolic gene cluster, which also encodes a putative invertase (invA), a regulatory protein (dhaR), an alcohol dehydrogenase (adhA), and an aldehyde dehydrogenase (aldA). The latter two enzymes may catalyze the oxidative conversion of n-butanol, the hydrolytic product of 1-chlorobutane, to n-butyric acid, a growth substrate for many bacteria. The activity of the dhaR gene product was analyzed in Pseudomonas sp. strain GJ1, in which it appeared to function as a repressor of dhaA expression. The 1,2-dibromoethane-degrading strain GP1 contained a conserved DNA segment of 2.7 kb, which included dhaR, dhaA, and part of invA. A 12-nucleotide deletion in dhaR led to constitutive expression of dhaA in strain GP1, in contrast to the inducible expression of dhaA in strain NCIMB13064. The 1, 3-dichloropropene-degrading strain 170 possessed a conserved DNA segment of 1.3 kb harboring little more than the coding region of the dhaA gene. In strains 170 and GP1, a putative integrase gene was found next to the conserved dhaA segment, which suggests that integration events were responsible for the acquisition of these DNA segments. The data indicate that horizontal gene transfer and integrase-dependent gene acquisition were the key mechanisms for the evolution of catabolic pathways for the man-made chemicals 1, 3-dichloropropene and 1,2-dibromoethane.

Haloalkane-Utilizing Rhodococcus Strains Isolated from Geographically Distinct Locations Possess a Highly Conserved Gene Cluster Encoding Haloalkane Catabolism

The sequences of the 16S rRNA and haloalkane dehalogenase (dhaA) genes of five gram-positive haloalkane-utilizing bacteria isolated from contaminated sites in Europe, Japan, and the United States and of the archetypal haloalkane-degrading bacterium Rhodococcus sp. strain NCIMB13064 were compared. The 16S rRNA gene sequences showed less than 1% sequence divergence, and all haloalkane degraders clearly belonged to the genus Rhodococcus. All strains shared a completely conserved dhaA gene, suggesting that the dhaA genes were recently derived from a common ancestor. The genetic organization of the dhaA gene region in each of the haloalkane degraders was examined by hybridization analysis and DNA sequencing. Three different groups could be defined on the basis of the extent of the conserved dhaA segment. The minimal structure present in all strains consisted of a conserved region of 12.5 kb, which included the haloalkane-degradative gene cluster that was previously found in strain NCIMB13064. Plasmids of different sizes were found in all strains. Southern hybridization analysis with a dhaA gene probe suggested that all haloalkane degraders carry the dhaA gene region both on the chromosome and on a plasmid (70 to 100 kb). This suggests that an ancestral plasmid was transferred between these Rhodococcus strains and subsequently has undergone insertions or deletions. In addition, transposition events and/or plasmid integration may be responsible for positioning the dhaA gene region on the chromosome. The data suggest that the haloalkane dehalogenase gene regions of these gram-positive haloalkane-utilizing bacteria are composed of a single catabolic gene cluster that was recently distributed worldwide.

The chemical versatility of the β–α–β fold: Catalytic promiscuity and divergent evolution in the tautomerase superfamily

Abstract.Tautomerase superfamily members have an amino-terminal proline and a β–α–β fold, and include 4-oxalocrotonate tautomerase (4-OT), 5-(carboxymethyl)-2-hydroxymuconate isomerase (CHMI), trans- and cis-3-chloroacrylic acid dehalogenase (CaaD and cis-CaaD, respectively), malonate semialdehyde decarboxylase (MSAD), and macrophage migration inhibitory factor (MIF), which exhibits a phenylpyruvate tautomerase (PPT) activity. Pro-1 is a base (4-OT, CHMI, the PPT activity of MIF) or an acid (CaaD, cis-CaaD, MSAD). Components of the catalytic machinery have been identified and mechanistic hypotheses formulated. Characterization of new homologues shows that these mechanisms are incomplete. 4-OT, CaaD, cis-CaaD, and MSAD also have promiscuous activities with a hydratase activity in CaaD, cis-CaaD, and MSAD, PPT activity in CaaD and cis-CaaD, and CaaD and cis-CaaD activities in 4-OT. The shared promiscuous activities provide evidence for divergent evolution from a common ancestor, give hints about mechanistic relationships, and implicate catalytic promiscuity in the emergence of new enzymes.

The Escherichia coli multidrug transporter MdfA catalyzes both electrogenic and electroneutral transport reactions

The resistance of cells to many drugs simultaneously (multidrug resistance) often involves the expression of membrane transporters (Mdrs); each recognizes and expels a broad spectrum of chemically unrelated drugs from the cell. The Escherichia coli Mdr transporter MdfA is able to transport differentially charged substrates in exchange for protons. This includes neutral compounds, namely chloramphenicol and thiamphenicol, and lipophilic cations such as tetraphenylphosphonium and ethidium. Here we show that the chloramphenicol and thiamphenicol transport reactions are electrogenic, whereas the transport of several monovalent cationic substrates is electroneutral. Therefore, unlike with positively charged substrates, the transmembrane electrical potential (negative inside) constitutes a major part of the driving force for the transport of electroneutral substrates by MdfA. These results demonstrate an unprecedented ability of a single secondary transporter to catalyze discrete transport reactions that differ in their electrogenicity and are governed by different components of the proton motive force.

Enzymatic Synthesis of Enantiopure α- and β-Amino Acids by Phenylalanine Aminomutase-Catalysed Amination of Cinnamic Acid Derivatives

The phenylalanine aminomutase (PAM) from Taxus chinensis catalyses the conversion of α‐phenylalanine to β‐phenylalanine, an important step in the biosynthesis of the N‐benzoyl phenylisoserinoyl side‐chain of the anticancer drug taxol. Mechanistic studies on PAM have suggested that (E)‐cinnamic acid is an intermediate in the mutase reaction and that it can be released from the enzymes active site. Here we describe a novel synthetic strategy that is based on the finding that ring‐substituted (E)‐cinnamic acids can serve as a substrate in PAM‐catalysed ammonia addition reactions for the biocatalytic production of several important β‐amino acids. The enzyme has a broad substrate range and a high enantioselectivity with cinnamic acid derivatives; this allows the synthesis of several non‐natural aromatic α‐ and β‐amino acids in excellent enantiomeric excess (ee >99 %). The internal 5‐methylene‐3,5‐dihydroimidazol‐4‐one (MIO) cofactor is essential for the PAM‐catalysed amination reactions. The regioselectivity of amination reactions was influenced by the nature of the ring substituent.

PA0305 of Pseudomonas aeruginosa is a quorum quenching acylhomoserine lactone acylase belonging to the Ntn hydrolase superfamily.

The Pseudomonas aeruginosa PAO1 genome has at least two genes, pvdQ and quiP, encoding acylhomoserine lactone (AHL) acylases. Two additional genes, pa1893 and pa0305, have been predicted to encode penicillin acylase proteins, but have not been characterized. Initial studies on a pa0305 transposon insertion mutant suggested that the gene is not related to the AHL growth phenotype of P. aeruginosa. The close similarity (67 %) of pa0305 to HacB, an AHL acylase of Pseudomonas syringae, prompted us to investigate whether the PA0305 protein might also function as an AHL acylase. The pa0305 gene has been cloned and the protein (PA0305) has been overproduced, purified and subjected to functional characterization. Analysis of the purified protein showed that, like β-lactam acylases, PA0305 undergoes post-translational processing resulting in α- and β-subunits, with the catalytic serine as the first amino acid of the β-subunit, strongly suggesting that PA0305 is a member of the N-terminal nucleophile hydrolase superfamily. Using a biosensor assay, PA0305his was shown to degrade AHLs with acyl side chains ranging in length from 6 to 14 carbons. Kinetics studies using N-octanoyl-L-homoserine lactone (C(8)-HSL) and N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C(12)-HSL) as substrates showed that the enzyme has a robust activity towards these two AHLs, with apparent K(cat)/K(m) values of 0.14 × 10(4) M(-1) s(-1) towards C(8)-HSL and 7.8 × 10(4) M(-1 )s(-1) towards 3-oxo-C(12)-HSL. Overexpression of the pa0305 gene in P. aeruginosa showed significant reductions in both accumulation of 3-oxo-C(12)-HSL and expression of virulence factors. A mutant P. aeruginosa strain with a deleted pa0305 gene showed a slightly increased capacity to kill Caenorhabditis elegans compared with the P. aeruginosa PAO1 wild-type strain and the PAO1 strain carrying a plasmid overexpressing pa0305. The harmful effects of the Δpa0305 strain on the animals were most visible at 5 days post-exposure and the mortality rate of the animals fed on the Δpa0305 strain was faster than for the animals fed on either the wild-type strain or the strain overexpressing pa0305. In conclusion, the pa0305 gene encodes an efficient acylase with activity towards long-chain homoserine lactones, including 3-oxo-C(12)-HSL, the natural quorum sensing signal molecule in P. aeruginosa, and we propose to name this acylase HacB.

Bridging between Organocatalysis and Biocatalysis: Asymmetric Addition of Acetaldehyde to β‐Nitrostyrenes Catalyzed by a Promiscuous Proline‐Based Tautomerase

In recent years, organocatalysis has become one of the main areas in asymmetric catalysis of carbon–carbon bond-forming reactions. The fast evolution of the organocatalysis field has been particularly fueled by aminocatalysis, in which secondary and primary amines react with carbonyl compounds to give enamine and iminium ion intermediates. The field was completely transformed during the last two decades by the seminal contributions of List, MacMillan, Yamaguchi, and co-workers. The natural chiral amino acid proline and derivatives thereof were found to be powerful organocatalysts. These secondary amines are applied in substoichiometric quantities and afford high product yields and enantioselectivities in fundamental carbon–carbon bond-forming reactions such as aldolizations, 2,3b] Michael additions, 4, 5] Mannich reactions, 6] and Knoevenagel condensations. Inspired by the versatile success of proline and its derivatives as organocatalysts, we examined whether the enzyme 4-oxalocrotonate tautomerase (4-OT), which carries a catalytic amino-terminal proline (Pro = P), might be suitable to promiscuously catalyze carbon–carbon bondforming reactions. Herein, we describe the discovery and characterization of two 4-OT-catalyzed asymmetric carbon– carbon bond-forming Michael-type addition reactions. Considering our reported 4-OT-catalyzed aldolizations, this work is a pivotal step forward towards our aim to bridge organocatalysis and biocatalysis by developing a new class of biocatalysts that use the powerful proline-based enamine mechanism of organocatalysts but that take advantage of the water solubility and relatively high catalytic rates available with enzymes. A few elegant studies on promiscuous enzyme-catalyzed carbon–carbon bond-forming Michael additions have been reported, but most of these reactions proceed in organic solvents and with moderate stereocontrol. 4-OT is a stable enzyme composed of six identical subunits of only 62 amino acid residues each. It belongs to the tautomerase superfamily, a group of homologous proteins that share a conserved catalytic amino-terminal proline and a characteristic b-a-b structural fold. 12] 4-OT takes part in a degradation pathway for aromatic hydrocarbons in Pseudomonas putida mt-2, where it catalyzes the tautomerization of 2-hydroxy-2,4-hexadienedioate (1) into 2-oxo-3-hexenedioate (2, Scheme 1). The key catalytic residues of 4-OTare Pro-1,

Recent developments in enzyme promiscuity for carbon-carbon bond-forming reactions

Numerous enzymes have been found to catalyze additional and completely different types of reactions relative to the natural activity they evolved for. This phenomenon, called catalytic promiscuity, has proven to be a fruitful guide for the development of novel biocatalysts for organic synthesis purposes. As such, enzymes have been identified with promiscuous catalytic activity for, one or more, eminent types of carbon-carbon bond-forming reactions like aldol couplings, Michael(-type) additions, Mannich reactions, Henry reactions, and Knoevenagel condensations. This review focuses on enzymes that promiscuously catalyze these reaction types and exhibit high enantioselectivities (in case chiral products are obtained).