Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A. Phylogenetic tree of 83 Eurotiale order fungal IMPDHs, including 62 IMPDH-As and 21 IMPDH-Bs constructed using BALi-Phy . Branches with ≥80% support are shown. Five major subclades are found: Byssochlamys and Talaromyces (olive green), Aspergillus (purple), Penicillium IMPDH-As (blue), IMPDH-Bs (red), and outgroups composed closely related Trichophyton , Microsporum , and Coccoidiodes. Bi-partition branch supports are listed to the left of their node of origin. PbreA, PnalA, PbreB and PnalB are highlighted to illustrate the incongruence of the IMPH-A and IMPDH-B phylogeny. Resurrected ancestors are denoted by the number along the branches. MPA producers are denoted with stars. Branch length scale is shown at the bottom. Abbreviations: Acan , Aspergillus candidus ; Apse , Aspergillus pseudoglaucus ; BsAF , Byssochlamys sp AF001B; Pani , Paecilomyces nivea ; Pant , Penicillium antarcticum ; Pbre , P. brevicompactum ; Pbra, P. brasilianum ; Pcam , P. camemberti ; Pcar, P. carneum ; Pcop, P. coprophilum ; Pdig, P. digitatum ; Pexp , P. expansum ; Pfre , P. freii ; Pgris , P. griseofulvum ; Pita , P. italicum ; Pnal , P. nalgiovense ; Pnor, P. nordicum ; Ppar, P. parvum ; Ppol, P. polonicum ; Proq , P. roqueforti ; PruW, P. rubens Wisconsin ; Psam , P. samsonianum ; Psol , P. solitum ; Pste , P. steckii ; Pvul , P. vulpinum . B-G. The values of K iapp for MPA ( B ), k cat ( C ), K IMP ( D ), K NAD ( E ), k cat /K IMP ( F ) and k cat /K NAD ( G ) are shown for IMPDH-Bs from producers (Prod, magenta) and nonproducers (Non, teal) and ancestors (Anc, colored as noted), as well as MPA-sensitive IMPDHs from the literature (MPA S , black, defined as K iapp ≤ 30 nM), See , and and and for data and parameters for each enzyme. **, p ≤ 0.01. **, p ≤ 0.01. B. IMPDH-Bs and Anc3-Anc6 are MPA-resistant while Anc1 and Anc2 are MPA S . C. The values of k cat are similar for IMPDH-Bs and MPA S enzymes. D. The values of K IMP are larger for IMPDH-Bs than MPA S enzymes. E. The values of K NAD are comparable among IMPDH-Bs and MPA S enzymes. F. The values of k cat /K IMP of MPA S IMPDHs are greater than IMPDH-Bs. Producer IMPDH-Bs tend to be more active than nonproducer enzymes (p = 0.18; p= 0.02 if the thermolabile Pani B value is omitted). G. The values of k cat /K NAD of MPA S IMPDHs are greater than nonproducer IMPDH-Bs.

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Construct

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: Enzyme concentrations are listed in the Methods section. Reactions were with fixed substrate concentrations as listed and at 25°C unless otherwise specified. The average and range of duplicate reactions are plotted. Data was analyzed in GraphPad Prism. The values of K IMP were determined by fits to the Michaelis-Menten equation, values of K NAD were determined by fits to Michealis-Menten or substrate inhibition equation as appropriate. The values of K iapp for MPA were determined as described in the Methods. CI, 95% confidence intervals. Values are listed in .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Inhibition

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A . Clustal Omega sequence alignment of ancestral IMPDHs . Ancestors listed in order of appearance in the tree in . First change at a given position denoted in yellow, second change in green, third change in magenta. B. AlphaFold model of Anc1 colored by pLDDT . Inset shows PAE. C. Anc1 is shown in purple ribbon, substitutions in Anc2-7 are shown colored as in the key. K + , pink ball. IMP, gray sticks. MPA, olive sticks. These ligands were positioned by aligning Anc1 with the C. griseus E-XMP*•MPA complex (PDB: 1JR1, ).

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Sequencing

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A. Timelines for the changes in resistance (values of K iapp for MPA and RVP) and catalytic efficiency ( k cat /K IMP and k cat /K NAD ) for the resurrected ancestors. B. Ancestor substitutions near the MPA binding site. The structure of Anc1 (purple) was predicted using AlphaFold2 ( ; ). Substitutions in Anc2-7 are shown colored as in the key in panel A . K + , pink ball; IMP, gray sticks; MPA, olive sticks. These ligands were positioned by aligning Anc1 with the C. griseus E-XMP*•MPA complex (PDB: 1JR1, ). C-E. Timelines for the changes in resistance (values of K iapp for MPA and RVP) and catalytic efficiency ( k cat /K IMP and k cat /K NAD ) for the ancestors and their extant descendants. MPA producers are magenta, nonproducers are teal, ancestors are colored as in the key in panel A .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: A. Linear free energy relationship between the values of k cat /K IMP and MPA resistance ( K iapp ). MPA S , K iapp ≤ 30 nM; MPA-moderately resistant (MPA M ), 1 μM ≥ K iapp > 30 nM; MPA R , K iapp > 1 μM. See Tables S1 and S2 for all values. B. Linear free energy relationship between k cat /K NAD and MPA resistance ( K iapp ). Key as in panel A . C. E•RVP mimics E-XMP*. The structures of E-XMP*•MPA (cornflower blue, PDB: 1JR1) and E•RVP•MAD (olive, PDB: 1NF7; MAD = mycophenolate adenine dinucleotide) are overlaid. E-XMP* is shown in cyan, MPA in spring green, RVP in magenta, and MAD in dark magenta. Figure rendered in UCSF Chimera .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques:

Journal: bioRxiv

Article Title: An activity-resistance tradeoff constrains enzyme evolution

doi: 10.64898/2026.01.19.700455

Figure Lengend Snippet: Enzyme concentrations are listed in Methods section. Reactions performed in duplicate at 0.5* K IMP and 1.8* K NAD and 1 mM RVP at 25°C unless otherwise noted. Anc6, Pani B and Pfre B were assayed at 18°C. Pcam B was assayed at 11.5°C. Gray denotes representative eukaryotic IMPDHs, magenta denotes enzymes from MPA producers, teal denotes enzymes from nonproducers and ancestors are colored as in - .

Article Snippet: NAD + was purchased from ThermoFisher, IMP from MP Biomedicals and MPA from Sigma.

Techniques: