An enzyme-coupled microplate assay for activity and inhibition of hmdUMP hydrolysis by DNPH1.

2'-Deoxynucleoside 5'-monophosphate N-glycosidase 1 (DNPH1) hydrolyzes the epigenetically modified nucleotide 5-hydroxymethyl 2'-deoxyuridine 5'-monophosphate (hmdUMP) derived from DNA metabolism. Published assays of DNPH1 activity are low throughput, use high concentrations of DNPH1, and have not incorporated or characterized reactivity with the natural substrate. We describe the enzymatic synthesis of hmdUMP from commercially available materials and define its steady-state kinetics with DNPH1 using a sensitive, two-pathway enzyme coupled assay. This continuous absorbance-based assay works in 96-well plate format using nearly 500-fold less DNPH1 than previous methods. With a Z prime value of 0.92, the assay is suitable for high-throughput assays, screening of DNPH1 inhibitors, or characterization of other deoxynucleotide monophosphate hydrolases.

Rational engineering of 2-deoxyribose-5-phosphate aldolases for the biosynthesis of (R)-1,3-butanediol.

Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of (R)-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from Bacillus halodurans, TM1559 from Thermotoga maritima, and DeoC from Escherichia coli The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr224 in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys155 and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in E. coli cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for in vitro and in vivo 1,3BDO biosynthesis.

Protein engineering of aldolase LbDERA for enhanced activity toward real substrates with a high-throughput screening method coupled with an aldehyde dehydrogenase.

A new high-throughput method for screening 2-deoxyribose-5-phosphate aldolase variants with a higher activity toward aldol reaction of unnatural aldehydes was established for the first time by coupling with an aldehyde dehydrogenase LeADH. The error-prone PCR and site-directed saturation mutagenesis libraries of aldolase LbDERA were constructed and screened using the high-throughput method. Two improved variants, LbDERAT29L and LbDERAF163Y, were identified and combined, giving a double mutant LbDERAT29L/F163Y which showed 7-fold higher activity than the native enzyme. The crystal structure of LbDERAT29L/163Y obtained by X-ray diffraction with 1.77 Å resolution revealed the structural changes responsible for the significant activity improvement.

Structural insight for substrate tolerance to 2-deoxyribose-5-phosphate aldolase from the pathogen Streptococcus suis.

2-deoxyribose-5-phosphate aldolase (DERA) is a class I aldolase that catalyzes aldol condensation of two aldehydes in the active site, which is particularly germane in drug manufacture. Structural and biochemical studies have shown that the active site of DERA is typically loosely packed and displays broader substrate specificity despite sharing conserved folding architecture with other aldolases. The most distinctive structural feature of DERA compared to other aldolases is short and flexible C-terminal region. This region is also responsible for substrate recognition. Therefore, substrate tolerance may be related to the C-terminal structural features of DERA. Here, we determined the crystal structures of full length and C-terminal truncated DERA from Streptococcus suis (SsDERA). In common, both contained the typical (α/ß)8 TIM-barrel fold of class I aldolases. Surprisingly, C-terminal truncation resulting in missing the last α9 and ß8 secondary elements, allowed DERA to maintain activity comparable to the fulllength enzyme. Specifically, Arg186 and Ser205 residues at the C-terminus appeared mutually supplemental or less indispensible for substrate phosphate moiety recognition. Our results suggest that DERA might adopt a shorter C-terminal region than conventional aldolases during evolution pathway, resulting in a broader range of substrate tolerance through active site flexibility.