Crystal structures of phosphite dehydrogenase provide insights into nicotinamide cofactor regeneration.

The enzyme phosphite dehydrogenase (PTDH) catalyzes the NAD(+)-dependent conversion of phosphite to phosphate and represents the first biological catalyst that has been shown to conduct the enzymatic oxidation of phosphorus. Despite investigation for more than a decade into both the mechanism of its unusual reaction and its utility in cofactor regeneration, there has been a lack of any structural data for PTDH. Here we present the cocrystal structure of an engineered thermostable variant of PTDH bound to NAD(+) (1.7 Å resolution), as well as four other cocrystal structures of thermostable PTDH and its variants with different ligands (all between 1.85 and 2.3 Å resolution). These structures provide a molecular framework for understanding prior mutational analysis and point to additional residues, located in the active site, that may contribute to the enzymatic activity of this highly unusual catalyst.

Efficient production of L-ribose with a recombinant Escherichia coli biocatalyst.

A new synthetic platform with potential for the production of several rare sugars, with l-ribose as the model target, is described. The gene encoding the unique NAD-dependent mannitol-1-dehydrogenase (MDH) from Apium graveolens (garden celery) was synthetically constructed for optimal expression in Escherichia coli. This MDH enzyme catalyzes the interconversion of several polyols and their l-sugar counterparts, including the conversion of ribitol to l-ribose. Expression of recombinant MDH in the active form was successfully achieved, and one-step purification was demonstrated. Using the created recombinant E. coli strain as a whole-cell catalyst, the synthetic utility was demonstrated for production of l-ribose, and the system was improved using shaken flask experiments. It was determined that addition of 50 to 500 microM ZnCl(2) and addition of 5 g/liter glycerol both improved production. The final levels of conversion achieved were >70% at a concentration of 40 g/liter and >50% at a concentration of 100 g/liter. The best conditions determined were then scaled up to a 1-liter fermentation that resulted in 55% conversion of 100 g/liter ribitol in 72 h, for a volumetric productivity of 17.4 g liter(-1) day(-1). This system represents a significantly improved method for the large-scale production of l-ribose.

Heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster.

Fosfomycin is a clinically utilized, highly effective antibiotic, which is active against methicillin- and vancomycin-resistant pathogens. Here we report the cloning and characterization of a complete fosfomycin biosynthetic cluster from Streptomyces fradiae and heterologous production of fosfomycin in S. lividans. Sequence analysis coupled with gene deletion and disruption revealed that the minimal cluster consists of fom1-4, fomA-D. A LuxR-type activator that was apparently required for heterologous fosfomycin production was also discovered approximately 13 kb away from the cluster and was named fomR. The genes fomE and fomF, previously thought to be involved in fosfomycin biosynthesis, were shown not to be essential by gene disruption. This work provides new insights into fosfomycin biosynthesis and opens the door for fosfomycin overproduction and creation of new analogs via biomolecular pathway engineering.

New insight into the mechanism of methyl transfer during the biosynthesis of fosfomycin.

Hydroxyethylphosphonate is a required intermediate in fosfomycin biosynthesis.

Optimizing a biocatalyst for improved NAD(P)H regeneration: directed evolution of phosphite dehydrogenase.

Cofactor regeneration is an important solution to the problem of implementing complex cofactor requiring enzymatic reactions at the industrial scale. NAD(P)H-dependent oxidoreductases are highly valuable biocatalysts, but the high cost of the nicotinamide cofactors necessitates in situ cofactor regeneration for preparative applications. Here we report the use of directed evolution to enhance the industrially important properties of phosphite dehydrogenase for NAD(P)H regeneration. A two-tiered sorting method of selection and screening was used in conjunction with random and rational mutagenesis. Following six rounds of directed evolution, soluble expression in E. coli was increased more than 3-fold, while the turnover rate was increased about 2-fold, effectively lowering the cost of the enzyme by >6-fold. Large-scale production of the final mutant enzyme by fermentation resulted in approximately 6-times higher yield (Units/Liter) than the WT enzyme. The enhancements of PTDH were independent of expression vector and E. coli strain utilized. The advantage of the final mutant over the WT enzyme was demonstrated using the industrially relevant bioconversion of trimethylpyruvate to L-tert-leucine. The mutations discovered are discussed in the context of a three dimensional structural model and the resulting changes in kinetics and soluble expression. The engineered phosphite dehydrogenase has great potential for NAD(P)H regeneration in industrial biocatalysis.

Mechanistic investigation of a highly active phosphite dehydrogenase mutant and its application for NADPH regeneration.

NAD(P)H regeneration is important for biocatalytic reactions that require these costly cofactors. A mutant phosphite dehydrogenase (PTDH-E175A/A176R) that utilizes both NAD and NADP efficiently is a very promising system for NAD(P)H regeneration. In this work, both the kinetic mechanism and practical application of PTDH-E175A/A176R were investigated for better understanding of the enzyme and to provide a basis for future optimization. Kinetic isotope effect studies with PTDH-E175A/A176R showed that the hydride transfer step is (partially) rate determining with both NAD and NADP giving (D)V values of 2.2 and 1.7, respectively, and (D)V/K(m,phosphite) values of 1.9 and 1.7, respectively. To better comprehend the relaxed cofactor specificity, the cofactor dissociation constants were determined utilizing tryptophan intrinsic fluorescence quenching. The dissociation constants of NAD and NADP with PTDH-E175A/A176R were 53 and 1.9 microm, respectively, while those of the products NADH and NADPH were 17.4 and 1.22 microm, respectively. Using sulfite as a substrate mimic, the binding order was established, with the cofactor binding first and sulfite binding second. The low dissociation constant for the cofactor product NADPH combined with the reduced values for (D)V and k(cat) implies that product release may become partially rate determining. However, product inhibition does not prevent efficient in situ NADPH regeneration by PTDH-E175A/A176R in a model system in which xylose was converted into xylitol by NADP-dependent xylose reductase. The in situ regeneration proceeded at a rate approximately fourfold faster with PTDH-E175A/A176R than with either WT PTDH or a NADP-specific Pseudomonas sp.101 formate dehydrogenase mutant with a total turnover number for NADPH of 2500.

Single-step bioconversion for the preparation of L-gulose and L-galactose.

Both carbohydrate monomers L-gulose and L-galactose are rarely found in nature, but are of great importance in pharmacy R&D and manufacturing. A method for the production of L-gulose and L-galactose is described that utilizes recombinant Escherichia coli harboring a unique mannitol dehydrogenase. The recombinant E. coli system was optimized by genetic manipulation and directed evolution of the recombinant protein to improve conversion. The resulting production process requires a single step, represents the first readily scalable system for the production of these sugars, is environmentally friendly, and utilizes inexpensive reagents, while producing L-galactose at 4.6 g L(-1)d(-1) and L-gulose at 0.90 g L(-1)d(-1).

Directed evolution toward improved production of L-ribose from ribitol.

Improvement of the one-step production of L-ribose from ribitol using a recombinant Escherichia coli is described. The gene encoding the enzyme mannitol-1-dehydrogenase (MDH) from Apium graveolens has previously been codon-optimized, cloned into the constitutive pZuc10 vector, and expressed in E. coli. This MDH catalyzes the NAD-dependent conversion of mannitol to D-mannose and has the ability to convert several polyols to their L-sugar counterparts, including ribitol to L-ribose. Here, three rounds of directed evolution using libraries generated through error-prone PCR and screened using a dinitrosalicylate reagent were prepared. Mutants were selected for improved conversion of L-ribose, and the best mutant was isolated by combining two round 2 mutations. Libraries were also selected for thermal stability and screened at increasingly higher temperatures with each round of mutagenesis. An overall 19.2-fold improvement was observed with a final conversion of 46.6 +/- 1.7% and a productivity of 3.88 +/- 0.14 gL(-1)d(-1) in 50 mL shaken flasks at 34 degrees C. Further characterization of the mutants suggests that increased enzyme thermal stability and expression are responsible for the increase in L-ribose production. The mutant E. coli production strain isolated represents an improved system for large-scale production of L-ribose.

Biosynthesis of 2-hydroxyethylphosphonate, an unexpected intermediate common to multiple phosphonate biosynthetic pathways.

Phosphonic acids encompass a common yet chemically diverse class of natural products that often possess potent biological activities. Here we report that, despite the significant structural differences among many of these compounds, their biosynthetic routes contain an unexpected common intermediate, 2-hydroxyethyl-phosphonate, which is synthesized from phosphonoacetaldehyde by a distinct family of metal-dependent alcohol dehydrogenases (ADHs). Although the sequence identity of the ADH family members is relatively low (34-37%), in vitro biochemical characterization of the homologs involved in biosynthesis of the antibiotics fosfomycin, phosphinothricin tripeptide, and dehydrophos (formerly A53868) unequivocally confirms their enzymatic activities. These unique ADHs have exquisite substrate specificity, unusual metal requirements, and an unprecedented monomeric quaternary structure. Further, sequence analysis shows that these ADHs form a monophyletic group along with additional family members encoded by putative phosphonate biosynthetic gene clusters. Thus, the reduction of phosphonoacetaldehyde to hydroxyethyl-phosphonate may represent a common step in the biosynthesis of many phosphonate natural products, a finding that lends insight into the evolution of phosphonate biosynthetic pathways and the chemical structures of new C-P containing secondary metabolites.