Development of a kinetic model and figures of merit for formaldehyde carboligations catalyzed by formolase enzymes

Abstract

There is an increasing interest in the upgrading of inexpensive and abundant C1 feedstocks to higher carbon products. Linear carbon ligation routes are of particular interest due to their simplicity and potential for high carbon efficiencies. The formolase (FLS) enzyme was computationally designed to catalyze the formose reaction, where formaldehyde molecules are coupled to produce a mixture of C2 (glycolaldehyde) and C3 (dihydroxyacetone) molecules. Recent protein engineering efforts have resulted in the introduction of several FLS variants with altered catalytic properties. As is often the case with enzymes catalyzing reactions with complex and/or nonnatural trajectories, there are no mechanistic kinetic models that fully describe the activity of the FLS enzyme. FLS variants are typically evaluated by fitting rate data to empirical rate laws, with some variation of the kcat/KM ratio used to report and rank performances. The apparent parameters estimated in this manner are unlikely to capture the full catalytic performance of these enzymes. In this study, we derive a mechanistic rate law describing FLS activity as well as theory‐based figures of merit to rank FLS performance under relevant conditions. We proceed to fit the rate equation to initial rate data obtained from several FLS mutants, and use the figures of merit to compare the mutations. This study provides a theoretical framework for comparing FLS enzymes which will be essential as novel carbon ligation pathways are devised and implemented.

Revealing reaction intermediates in one-carbon elongation ThDP/CoA dependent enzyme family

“…Recently, the importance of HACL/S in microbial and biochemical conversion of one-carbon (C1) compounds to value-added products has been highlighted, leveraging the wide substrate specificity range of the enzyme for iterative C1 elongation to yield a variety of small molecules 7 . Additionally, HACL/S-based pathways are favored for synthetic biology applications due to their superior kinetic parameters compared to C1 assimilation by other ThDP-dependent carboligases 6,9 . Although HACL/Ss have been the focus of several biochemical studies, and some structural analysis was performed based on homology-guided modeling, most studies have centered on the acyloin condensation reaction between formaldehyde and fCoA (C1-C1 condensation) [4][5][6] .…”

Identification of 2-Hydroxyacyl-CoA Synthases with High Acyloin Condensation Activity for Orthogonal One-Carbon Bioconversion

“…To address these issues, researchers developed different synthetic metabolic pathways, which are built upon newly discovered or engineered C1–C1 coupling enzymes, such as formolase (FLS) and associated variants − and 2-hydroxyacyl-CoA synthase (HACS). , However, because these enzymes often exploit a natural substrate promiscuity, many of them suffer from suboptimal kinetic parameters, even after extensive engineering efforts . For example, the engineered FLS that catalyzes C–C coupling of two or three formaldehyde molecules into glycolaldehyde or dihydroxyacetone (Figure ) demonstrated a turnover frequency ( k cat ) of only up to 1.5 s –1 and apparent Michaelis–Menten constants ( K M ) for formaldehyde exceeding 100 mM, even after several rounds of directed evolution. − , Considering the high toxicity of formaldehyde, , these enzymes are not suitable for use not only in actively growing cultures but also in resting cells. Efforts to substantially improve affinity of FLS toward formaldehyde ( K M = 18 mM) led to an extremely poor turnover ( k cat = 0.1 s –1 ), which requires high cell density (protein loading) to demonstrate product synthesis at the desired rate …”

“…26 For example, the engineered FLS that catalyzes C−C coupling of two or three formaldehyde molecules into glycolaldehyde or dihydroxyacetone (Figure 1) demonstrated a turnover frequency (k cat ) of only up to 1.5 s −1 and apparent Michaelis−Menten constants (K M ) for formaldehyde exceeding 100 mM, even after several rounds of directed evolution. [20][21][22][23]27 Considering the high toxicity of formaldehyde, 11,28 these enzymes are not suitable for use not only in actively growing cultures but also in resting cells. Efforts to substantially improve affinity of FLS toward formaldehyde (K M = 18 mM) led to an extremely poor turnover (k cat = 0.1 s −1 ), which requires high cell density (protein loading) to demonstrate product synthesis at the desired rate.…”