Hélène Bergeron

Crystal structures of cyclohexanone monooxygenase reveal complex domain movements and a sliding cofactor

Cyclohexanone monooxygenase (CHMO) is a flavoprotein that carries out the archetypical Baeyer-Villiger oxidation of a variety of cyclic ketones into lactones. Using NADPH and O(2) as cosubstrates, the enzyme inserts one atom of oxygen into the substrate in a complex catalytic mechanism that involves the formation of a flavin-peroxide and Criegee intermediate. We present here the atomic structures of CHMO from an environmental Rhodococcus strain bound with FAD and NADP(+) in two distinct states, to resolutions of 2.3 and 2.2 A. The two conformations reveal domain shifts around multiple linkers and loop movements, involving conserved arginine 329 and tryptophan 492, which effect a translation of the nicotinamide resulting in a sliding cofactor. Consequently, the cofactor is ideally situated and subsequently repositioned during the catalytic cycle to first reduce the flavin and later stabilize formation of the Criegee intermediate. Concurrent movements of a loop adjacent to the active site demonstrate how this protein can effect large changes in the size and shape of the substrate binding pocket to accommodate a diverse range of substrates. Finally, the previously identified BVMO signature sequence is highlighted for its role in coordinating domain movements. Taken together, these structures provide mechanistic insights into CHMO-catalyzed Baeyer-Villiger oxidation.

Identification of a membrane protein and a truncated LysR-type regulator associated with the toluene degradation pathway in Pseudomonas putida F1

A 3 kb DNA region upstream of the toluene degradation (tod) genes, todFC1C2BADEGIH, in Pseudomonas putida F1 (PpF1) was sequenced. Two divergently arranged open reading frames, todR and todX, were identified. A toluene-inducible promoter was localized in front of todX, and the transcription start point was mapped. This promoter is probably responsible for the expression of all tod structural genes. TodX was found to be a membrane protein. Its predicted amino acid sequence (453 residues; Mr 48265) exhibits considerable similarity with the FadL protein of Escherichia coli, an outer membrane protein required for binding and transport of long-chain fatty acids. An apparent function of TodX is likely to be involved in facilitating the delivery of exogenous toluene inside the PpF1 cells. The sequence of TodR (100 residues) exhibits extensive homology with the DNA-binding domain of transcriptional activators of the LysR family, but todR was found to have a negligible role in tod gene regulation.

Pseudomonad cyclopentadecanone monooxygenase displaying an uncommon spectrum of Baeyer-Villiger oxidations of cyclic ketones.

ABSTRACT Baeyer-Villiger monooxygenases (BVMOs) are biocatalysts that offer the prospect of high chemo-, regio-, and enantioselectivity in the organic synthesis of lactones or esters from a variety of ketones. In this study, we have cloned, sequenced, and overexpressed in Escherichia coli a new BVMO, cyclopentadecanone monooxygenase (CpdB or CPDMO), originally derived from Pseudomonas sp. strain HI-70. The 601-residue primary structure of CpdB revealed only 29% to 50% sequence identity to those of known BVMOs. A new sequence motif, characterized by a cluster of charged residues, was identified in a subset of BVMO sequences that contain an N-terminal extension of ∼60 to 147 amino acids. The 64-kDa CPDMO enzyme was purified to apparent homogeneity, providing a specific activity of 3.94 μmol/min/mg protein and a 20% yield. CPDMO is monomeric and NADPH dependent and contains ∼1 mol flavin adenine dinucleotide per mole of protein. A deletion mutant suggested the importance of the N-terminal 54 amino acids to CPDMO activity. In addition, a Ser261Ala substitution in a Rossmann fold motif resulted in an improved stability and increased affinity of the enzyme towards NADPH compared to the wild-type enzyme (Km = 8 μM versus Km = 24 μM). Substrate profiling indicated that CPDMO is unusual among known BVMOs in being able to accommodate and oxidize both large and small ring substrates that include C11 to C15 ketones, methyl-substituted C5 and C6 ketones, and bicyclic ketones, such as decalone and β-tetralone. CPDMO has the highest affinity (Km = 5.8 μM) and the highest catalytic efficiency (kcat/Km ratio of 7.2 × 105 M−1 s−1) toward cyclopentadecanone, hence the Cpd designation. A number of whole-cell biotransformations were carried out, and as a result, CPDMO was found to have an excellent enantioselectivity (E > 200) as well as 99% S-selectivity toward 2-methylcyclohexanone for the production of 7-methyl-2-oxepanone, a potentially valuable chiral building block. Although showing a modest selectivity (E = 5.8), macrolactone formation of 15-hexadecanolide from the kinetic resolution of 2-methylcyclopentadecanone using CPDMO was also demonstrated.

Improvement of the Thermostability and Activity of a Pectate Lyase by Single Amino Acid Substitutions, Using a Strategy Based on Melting-Temperature-Guided Sequence Alignment

ABSTRACT In the vast number of random mutagenesis experiments that have targeted protein thermostability, single amino acid substitutions that increase the apparent melting temperature (Tm) of the enzyme more than 1 to 2°C are rare and often require the creation of a large library of mutated genes. Here we present a case where a single beneficial mutation (R236F) of a hemp fiber-processing pectate lyase of Xanthomonas campestris origin (PLXc) produced a 6°C increase in Tm and a 23-fold increase in the half-life at 45°C without compromising the enzymes catalytic efficiency. This success was based on a variation of sequence alignment strategy where a mesophilic amino acid sequence is matched with the sequences of its thermophilic counterparts that have established Tm values. Altogether, two-thirds of the nine targeted single amino acid substitutions were found to have effects either on the thermostability or on the catalytic activity of the enzyme, evidence of a high success rate of mutation without the creation of a large gene library and subsequent screening of clones. Combination of R236F with another beneficial mutation (A31G) resulted in at least a twofold increase in specific activity while preserving the improved Tm value. To understand the structural basis for the increased thermal stability or activity, the variant R236F and A31G R236F proteins and wild-type PLXc were purified and crystallized. By structure analysis and computational methods, hydrophobic desolvation was found to be the driving force for the increased stability with R236F.

Sequence and expression of the todGIH genes involved in the last three steps of toluene degradation by Pseudomonas putida F1.

The todFC1C2BADE gene cluster in Pseudomonas putida F1 encodes enzymes for the first four steps of toluene degradation, leading to the formation of 2-hydroxypenta-2,4-dienoate (HPD). Here, we report the nucleotide (nt) sequence and expression of the remaining three genes of the tod pathway, downstream from todE and arranged in the order, todGIH. The deduced amino acid (aa) sequences of TodG [HPD hydratase (268 aa)], TodH [4-hydroxy-2-oxovalerate (HO) aldolase (352 aa)] and TodI [acylating aldehyde (AA) dehydrogenase (316 aa)] are compared with the isofunctional proteins present in the meta-cleavage pathways of other bacteria. New sequence motifs are identified. The highly conserved TodH and TodI sequences are potentially useful DNA probes for biomonitoring purposes.

Sequence and expression of the bpdC1C2BADE genes involved in the initial steps of biphenyl/chlorobiphenyl degradation by Rhodococcus sp. M5

The nucleotide (nt) sequence of the bpdC1C2BADE genes which encode the first three enzymes in the biphenyl (BP) degradation pathway of Gram+ Rhodococcus sp. M5 (formerly Arthrobacter M5) was determined. Except for the ferredoxin component (BpdB) of the initial BP dioxygenase, the predicted amino acid (aa) sequences of the remaining proteins are found to be more closely related to the counterpart proteins (TodC1C2BADE) present in the toluene-degrader, Pseudomonas putida F1, than those of three BP-degrading pseudomonads. The cloned bpd genes were verified by their expression in the Escherichia coli T7 RNA polymerase/promoter system. In E. coli, BpdA was able to complement TodC1C2B in indigo biosynthesis, although the M5 native or cloned BP dioxygenase does not carry out this reaction.

Cloning, Baeyer-Villiger Biooxidations, and Structures of the Camphor Pathway 2-Oxo-Δ3-4,5,5-Trimethylcyclopentenylacetyl-Coenzyme A Monooxygenase of Pseudomonas putida ATCC 17453

ABSTRACT A dimeric Baeyer-Villiger monooxygenase (BVMO) catalyzing the lactonization of 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-coenzyme A (CoA), a key intermediate in the metabolism of camphor by Pseudomonas putida ATCC 17453, had been initially characterized in 1983 by Ougham and coworkers (H. J. Ougham, D. G. Taylor, and P. W. Trudgill, J. Bacteriol. 153:140–152, 1983). Here we cloned and overexpressed the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetyl-CoA monooxygenase (OTEMO) in Escherichia coli and determined its three-dimensional structure with bound flavin adenine dinucleotide (FAD) at a 1.95-Å resolution as well as with bound FAD and NADP+ at a 2.0-Å resolution. OTEMO represents the first homodimeric type 1 BVMO structure bound to FAD/NADP+. A comparison of several crystal forms of OTEMO bound to FAD and NADP+ revealed a conformational plasticity of several loop regions, some of which have been implicated in contributing to the substrate specificity profile of structurally related BVMOs. Substrate specificity studies confirmed that the 2-oxo-Δ3-4,5,5-trimethylcyclopentenylacetic acid coenzyme A ester is preferred over the free acid. However, the catalytic efficiency (k cat/Km ) favors 2-n-hexyl cyclopentanone (4.3 × 105 M−1 s−1) as a substrate, although its affinity (Km = 32 μM) was lower than that of the CoA-activated substrate (Km = 18 μM). In whole-cell biotransformation experiments, OTEMO showed a unique enantiocomplementarity to the action of the prototypical cyclohexanone monooxygenase (CHMO) and appeared to be particularly useful for the oxidation of 4-substituted cyclohexanones. Overall, this work extends our understanding of the molecular structure and mechanistic complexity of the type 1 family of BVMOs and expands the catalytic repertoire of one of its original members.

Camphor Pathway Redux: Functional Recombinant Expression of 2,5- and 3,6-Diketocamphane Monooxygenases of Pseudomonas putida ATCC 17453 with Their Cognate Flavin Reductase Catalyzing Baeyer-Villiger Reactions

ABSTRACT Whereas the biochemical properties of the monooxygenase components that catalyze the oxidation of 2,5-diketocamphane and 3,6-diketocamphane (2,5-DKCMO and 3,6-DKCMO, respectively) in the initial catabolic steps of (+) and (−) isomeric forms of camphor (CAM) metabolism in Pseudomonas putida ATCC 17453 are relatively well characterized, the actual identity of the flavin reductase (Fred) component that provides the reduced flavin to the oxygenases has hitherto been ill defined. In this study, a 37-kDa Fred was purified from a camphor-induced culture of P. putida ATCC 17453 and this facilitated cloning and characterization of the requisite protein. The active Fred is a homodimer with a subunit molecular weight of 18,000 that uses NADH as an electron donor (K m = 32 μM), and it catalyzes the reduction of flavin mononucleotide (FMN) (K m = 3.6 μM; k cat = 283 s−1) in preference to flavin adenine dinucleotide (FAD) (K m = 19 μM; k cat = 128 s−1). Sequence determination of ∼40 kb of the CAM degradation plasmid revealed the locations of two isofunctional 2,5-DKCMO genes (camE 25–1 for 2,5-DKCMO-1 and camE 25–2 for 2,5-DKCMO-2) as well as that of a 3,6-DKCMO-encoding gene (camE 36). In addition, by pulsed-field gel electrophoresis, the CAM plasmid was established to be linear and ∼533 kb in length. To enable functional assessment of the two-component monooxygenase system in Baeyer-Villiger oxidations, recombinant plasmids expressing Fred in tandem with the respective 2,5-DKCMO- and 3,6-DKCMO-encoding genes in Escherichia coli were constructed. Comparative substrate profiling of the isofunctional 2,5-DCKMOs did not yield obvious differences in Baeyer-Villiger biooxidations, but they are distinct from 3,6-DKCMO in the stereoselective oxygenations with various mono- and bicyclic ketone substrates.

Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensis

There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)‐2‐benzyloxy‐propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3–5°C increase in the apparent melting temperature (Tm) of the mutants over the native BcEST that has a Tm of 50°C was outperformed by TtEST, a naturally occurring homologue with a Tm of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the α/β‐fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis.

Nocardia iowensis sp. nov., an organism rich in biocatalytically important enzymes and nitric oxide synthase

Nocardia strain NRRL 5646, isolated from a garden soil sample in Osceola, Iowa, USA, was initially of interest as an antibiotic producer. It contained biocatalytically important enzymes and represented the first described nitric oxide synthase enzyme system in bacteria. The present polyphasic taxonomic study was undertaken to differentiate strain NRRL 5646(T) from related species of the genus Nocardia. Chemotaxonomic analyses included determinations of the fatty acid methyl ester profile (C(16 : 1)omega6c/C(16 : 1)omega7c, C(16 : 0), C(18 : 1)omega9c and C(18 : 0) 10-methyl as major components), quinone [cyclo MK-8(H(4)) as the major component], polar lipid (diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and phosphatidylinositol mannoside as major components) and mycolic acid. These results supported its placement within the genus Nocardia. Biochemical testing and 16S rRNA, 65-kDa heat-shock protein (hsp65) and preprotein translocase (secA1) gene sequence analyses differentiated strain NRRL 5646(T) from recognized Nocardia species. Previous studies have demonstrated that other genetic sequences (carboxylic acid reductase, Nocardia phosphopantetheinyl transferase and GTP cyclohydrolase I) from strain NRRL 5646(T) can also be used to substantiate its uniqueness. The level of 16S rRNA gene sequence similarity between strain NRRL 5646(T) and the type strains of Nocardia tenerifensis and Nocardia brasiliensis was 98.8 %. However, strain NRRL 5646(T) could be clearly distinguished from these Nocardia species based on DNA-DNA hybridization data. Consequently, strain NRRL 5646(T) is considered to represent a novel species of the genus Nocardia, for which the name Nocardia iowensis sp. nov. is proposed. The type strain is NRRL 5646(T) (=UI 122540(T)=NRRL B-24671(T)=DSM 45197(T)).