Biology subjects

Helmer, C. P. O.

Publications and source records attributed to Helmer, C. P. O..

3 recordsLinked to original sources

bioRxiv2026

Molecular mechanism of albicidin-dependent transcription activation by the antibiotic resistance factor AlbA

The escalating spread of antimicrobial resistance demands antibiotics with novel mechanisms of action. Albicidin is a potent DNA gyrase inhibitor produced by Xanthomonas species, and bacteria counter its activity through resistance mechanisms including the albicidin-sequestering protein AlbA from Klebsiella oxytoca. AlbA is a MerR family transcription activator that binds albicidin and stimulates expression of the albA gene, but how ligand recognition is coupled to transcription activation has remained unclear. Here, we use cryogenic electron microscopy and complementary biochemical assays to define the mechanism of AlbA-dependent transcription activation. Structures of transcription complexes reveal that AlbA remodels the promoter, repositioning the -35 element for productive sigma70 recognition and RNA polymerase engagement. Albicidin does not measurably alter AlbAs DNA affinity or the architecture of the mature open complex. Instead, transcription kinetics and experiments bypassing promoter opening indicate that albicidin selectively facilitates a promoter opening-dependent transition. AlbA is released upon synthesis of approximately five nucleotides, concomitant with downstream DNA melting, bubble translocation and upstream DNA rewinding rather than DNA scrunching. Together, these findings establish a sequential mechanism in which AlbA remodels promoter architecture, while albicidin tunes the kinetic transition to promoter opening, linking antibiotic recognition to induction of its sequestration machinery.

biochemistry↗

bioRxiv2025

Deciphering the evolutionary origin of the stereoselectivity of short-chain dehydrogenases in the oxidation of the monoterpenol 1-borneol

Enzyme engineering has produced numerous methods to optimize enzymes for biotechnological processes; however, less is known about how natural evolution creates new functionalities. We investigate the evolutionary emergence of enantioselectivity in plant borneol dehydrogenases (BDHs), which feature hydrophobic active-sites and are enantioselective towards dibornane-type monoterpenols. Ancestral sequence reconstruction provided a trajectory from the oldest unselective BDH ancestor N30 (E=12) toward a more recent selective ancestor N32, involving 19 mutations: 18 mutations are peripheral, one (I111L) occurs in the active-site. The mutation L111I in the hydrophobic pocket increased the selectivity of N30, while the back-mutation I111L decreased the selectivity of N32. Additional peripheral mutations (V136L/G169A/V183I) were required for high selectivity. Crystal structures suggested that protein dynamics, rather than structural changes shape these catalytic properties; this was confirmed by ML/MM simulations of ligand binding. Funnel-metadynamics simulations revealed a correlation between the active-sites solvent-accessible surface area (SASA) and selectivity. This potential evolutionary pathway shapes enantioselectivity, and guides future enzyme engineering campaigns.

biochemistry↗

bioRxiv2021

Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 [A] resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

biophysics↗