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.

Deciphering the role of recurrent FAD-dependent enzymes in bacterial phosphonate catabolism.

Phosphonates-compounds containing a direct C-P bond-represent an important source of phosphorus in some environments. The most common natural phosphonate is 2-aminoethylphosphonate (AEP). Many bacteria can break AEP down through specialized "hydrolytic" pathways, which start with the conversion of AEP into phosphonoacetaldehyde (PAA), catalyzed by the transaminase PhnW. However, the substrate scope of these pathways is very narrow, as PhnW cannot process other common AEP-related phosphonates, notably N-methyl AEP (M1AEP). Here, we describe a heterogeneous group of FAD-dependent oxidoreductases that efficiently oxidize M1AEP to directly generate PAA, thus expanding the versatility and usefulness of the hydrolytic AEP degradation pathways. Furthermore, some of these enzymes can also efficiently oxidize plain AEP. By doing so, they surrogate the role of PhnW in organisms that do not possess the transaminase and create novel versions of the AEP degradation pathways in which PAA is generated solely by oxidative deamination.

The Microbial Degradation of Natural and Anthropogenic Phosphonates.

Phosphonates are compounds containing a direct carbon-phosphorus (C-P) bond, which is particularly resistant to chemical and enzymatic degradation. They are environmentally ubiquitous: some of them are produced by microorganisms and invertebrates, whereas others derive from anthropogenic activities. Because of their chemical stability and potential toxicity, man-made phosphonates pose pollution problems, and many studies have tried to identify biocompatible systems for their elimination. On the other hand, phosphonates are a resource for microorganisms living in environments where the availability of phosphate is limited; thus, bacteria in particular have evolved systems to uptake and catabolize phosphonates. Such systems can be either selective for a narrow subset of compounds or show a broader specificity. The role, distribution, and evolution of microbial genes and enzymes dedicated to phosphonate degradation, as well as their regulation, have been the subjects of substantial studies. At least three enzyme systems have been identified so far, schematically distinguished based on the mechanism by which the C-P bond is ultimately cleaved-i.e., through either a hydrolytic, radical, or oxidative reaction. This review summarizes our current understanding of the molecular systems and pathways that serve to catabolize phosphonates, as well as the regulatory mechanisms that govern their activity.

L-serine biosynthesis in the human central nervous system: Structure and function of phosphoserine aminotransferase.

Organisms from all kingdoms of life synthesize L-serine (L-Ser) from 3-phosphoglycerate through the phosphorylated pathway, a three-step diversion of glycolysis. Phosphoserine aminotransferase (PSAT) catalyzes the intermediate step, the pyridoxal 5'-phosphate-dependent transamination of 3-phosphohydroxypyruvate and L-glutamate to O-phosphoserine (OPS) and α-ketoglutarate. PSAT is particularly relevant in the central nervous system of mammals because L-Ser is the metabolic precursor of D-serine, cysteine, phospholipids, and nucleotides. Several mutations in the human psat gene have been linked to serine deficiency disorders, characterized by severe neurological symptoms. Furthermore, PSAT is overexpressed in many tumors and this overexpression has been associated with poor clinical outcomes. Here, we report the detailed functional and structural characterization of the recombinant human PSAT. The reaction catalyzed by PSAT is reversible, with an equilibrium constant of about 10, and the enzyme is very efficient, with a kcat /Km of 5.9 × 106  M-1  s-1 , thus contributing in driving the pathway towards the products despite the extremely unfavorable first step catalyzed by 3-phosphoglycerate dehydrogenase. The 3D X-ray crystal structure of PSAT was solved in the substrate-free as well as in the OPS-bound forms. Both structures contain eight protein molecules in the asymmetric unit, arranged in four dimers, with a bound cofactor in each subunit. In the substrate-free form, the active site of PSAT contains a sulfate ion that, in the substrate-bound form, is replaced by the phosphate group of OPS. Interestingly, fast crystal soaking used to produce the substrate-bound form allowed the trapping of different intermediates along the catalytic cycle.

Human cytosolic transaminases: side activities and patterns of discrimination towards physiologically available alternative substrates.

Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow-a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.

A Novel Assay for Phosphoserine Phosphatase Exploiting Serine Acetyltransferase as the Coupling Enzyme.

Phosphoserine phosphatase (PSP) catalyzes the final step of de novo L-serine biosynthesis-the hydrolysis of phosphoserine to serine and inorganic phosphate-in humans, bacteria, and plants. In published works, the reaction is typically monitored through the discontinuous malachite green phosphate assay or, more rarely, through a continuous assay that couples phosphate release to the phosphorolysis of a chromogenic nucleoside by the enzyme purine nucleoside phosphorylase (PNP). These assays suffer from numerous drawbacks, and both rely on the detection of phosphate. We describe a new continuous assay that monitors the release of serine by exploiting bacterial serine acetyltransferase (SAT) as a reporter enzyme. SAT acetylates serine, consuming acetyl-CoA and releasing CoA-SH. CoA-SH spontaneously reacts with Ellman's reagent to produce a chromophore that absorbs light at 412 nm. The catalytic parameters estimated through the SAT-coupled assay are fully consistent with those obtained with the published methods, but the new assay exhibits several advantages. Particularly, it depletes L-serine, thus allowing more prolonged linearity in the kinetics. Moreover, as the SAT-coupled assay does not rely on phosphate detection, it can be used to investigate the inhibitory effect of phosphate on PSP.

Biochemical and Biophysical Characterization of Recombinant Human 3-Phosphoglycerate Dehydrogenase.

The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders.

Discovery of a New, Recurrent Enzyme in Bacterial Phosphonate Degradation: (R)-1-Hydroxy-2-aminoethylphosphonate Ammonia-lyase.

Phosphonates represent an important source of bioavailable phosphorus in certain environments. Accordingly, many microorganisms (particularly marine bacteria) possess catabolic pathways to degrade these molecules. One example is the widespread hydrolytic route for the breakdown of 2-aminoethylphosphonate (AEP, the most common biogenic phosphonate). In this pathway, the aminotransferase PhnW initially converts AEP into phosphonoacetaldehyde (PAA), which is then cleaved by the hydrolase PhnX to yield acetaldehyde and phosphate. This work focuses on a pyridoxal 5'-phosphate-dependent enzyme that is encoded in >13% of the bacterial gene clusters containing the phnW-phnX combination. This enzyme (which we termed PbfA) is annotated as a transaminase, but there is no obvious need for an additional transamination reaction in the established AEP degradation pathway. We report here that PbfA from the marine bacterium Vibrio splendidus catalyzes an elimination reaction on the naturally occurring compound (R)-1-hydroxy-2-aminoethylphosphonate (R-HAEP). The reaction releases ammonia and generates PAA, which can be then hydrolyzed by PhnX. In contrast, PbfA is not active toward the S enantiomer of HAEP or other HAEP-related compounds such as ethanolamine and d,l-isoserine, indicating a very high substrate specificity. We also show that R-HAEP (despite being structurally similar to AEP) is not processed efficiently by the PhnW-PhnX couple in the absence of PbfA. In summary, the reaction catalyzed by PbfA serves to funnel R-HAEP into the hydrolytic pathway for AEP degradation, expanding the scope and the usefulness of the pathway itself.

Off to a slow start: Analyzing lag phases and accelerating rates in steady-state enzyme kinetics.

Steady-state enzyme kinetics typically relies on the measurement of 'initial rates', obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or 'lag phase'. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s-1.