One substrate many enzymes virtual screening uncovers missing genes of carnitine biosynthesis in human and mouse.

The increasing availability of experimental and computational protein structures entices their use for function prediction. Here we develop an automated procedure to identify enzymes involved in metabolic reactions by assessing substrate conformations docked to a library of protein structures. By screening AlphaFold-modeled vitamin B6-dependent enzymes, we find that a metric based on catalytically favorable conformations at the enzyme active site performs best (AUROC Score=0.84) in identifying genes associated with known reactions. Applying this procedure, we identify the mammalian gene encoding hydroxytrimethyllysine aldolase (HTMLA), the second enzyme of carnitine biosynthesis. Upon experimental validation, we find that the top-ranked candidates, serine hydroxymethyl transferase (SHMT) 1 and 2, catalyze the HTMLA reaction. However, a mouse protein absent in humans (threonine aldolase; Tha1) catalyzes the reaction more efficiently. Tha1 did not rank highest based on the AlphaFold model, but its rank improved to second place using the experimental crystal structure we determined at 2.26 Å resolution. Our findings suggest that humans have lost a gene involved in carnitine biosynthesis, with HTMLA activity of SHMT partially compensating for its function.

A new inactive conformation of SARS-CoV-2 main protease.

The SARS-CoV-2 main protease (Mpro) has a pivotal role in mediating viral genome replication and transcription of the coronavirus, making it a promising target for drugs against the COVID-19 pandemic. Here, a crystal structure is presented in which Mpro adopts an inactive state that has never been observed before, called new-inactive. It is shown that the oxyanion loop, which is involved in substrate recognition and enzymatic activity, adopts a new catalytically incompetent conformation and that many of the key interactions of the active conformation of the enzyme around the active site are lost. Solvation/desolvation energetic contributions play an important role in the transition from the inactive to the active state, with Phe140 moving from an exposed to a buried environment and Asn142 moving from a buried environment to an exposed environment. In new-inactive Mpro a new cavity is present near the S2' subsite, and the N-terminal and C-terminal tails, as well as the dimeric interface, are perturbed, with partial destabilization of the dimeric assembly. This novel conformation is relevant both for comprehension of the mechanism of action of Mpro within the catalytic cycle and for the successful structure-based drug design of antiviral drugs.

Spectrophotometric methods for the measurement of soil pH: A reappraisal.

The pH of aqueous soil extracts is generally measured potentiometrically by glass electrode (GE). Spectrophotometric methods have also been used till around the '80s, and then they have apparently been abandoned. However, the use of microplates and spectrophotometers able to read absorbance values in them can significantly increase the speed of the data collection (saving analysis time), thus justifying their reappraisal. Three spectrophotometric methods are proposed in this work: a one-indicator (OISM), a separated three-indicator (STISM) and a mixed three-indicator (MTISM) spectrophotometric method. They are based on the addition of one or three colorimetric indicators (methyl red, bromocresol violet, and bromothymol blue) to the aqueous extract. The pH is measured through its absorption properties in the visible region. The analysis of 60 soil samples showed that STISM and MTISM results correlate well with the ones obtained by GE. The STISM method, being more simple and general than the other two methods, is proposed for quick routine analyses. The repeatability, reproducibility and accuracy of STISM (and of GE, for comparison) were evaluated by measuring several times the pH of a series of soil samples and of a certified reference soil. The standard deviations of STISM results were slightly worse than those of GE, whereas the accuracy was slightly better, indicating that STISM and GE have overall similar performances. STISM method is much faster than GE one: the analysis time saving is around 2 min per sample, and it becomes very high when hundreds of samples have to be analysed. It follows that STISM can represent an advantageous alternative to GE for rapid and accurate soil pH measurements.