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Michael addition reactions are highly useful in organic synthesis and are commonly accomplished using organocatalysts. However, the corresponding biocatalytic Michael additions are rare, typically lack synthetically useful substrate scope, and suffer from low stereoselectivity. Herein we report a biocatalytic nitro-Michael addition, catalyzed by NahE, that proceeds with low catalyst loading at room temperature in moderate to excellent enantioselectivity and high yields. A series of β-nitrostyrenes reacted with pyruvate in the presence of NahE to give, after oxidative decarboxylation, β-aryl-γ-nitrobutyric acids in up to 99 % yield without need for chromatography, providing a simple preparative-scale route to chiral GABA analogues. This reaction represents the first example of an aldolase displaying promiscuous Michaelase activity and opens the use of nitroalkenes in place of aldehydes as substrates for aldolases., (© 2022 Wiley-VCH GmbH.)

Phosphate ester analogs in which the bridging oxygen is replaced with a methylene or fluoromethylene group are well known non-hydrolyzable mimics of use as inhibitors and substrate analogs for reactions involving phosphate esters. Properties of the replaced oxygen are often best mimicked by a mono-fluoromethylene group, but such groups are challenging to synthesize and can exist as two stereoisomers. Here, we describe the protocol for our method of synthesizing the α-fluoromethylene analogs of d-glucose 6-phosphate (G6P), as well as the methylene and difluoromethylene analogs, and their application in the study of 1l-myo-inositol-1-phosphate synthase (mIPS). mIPS catalyzes the synthesis of 1l-myo-inositol 1-phosphate (mI1P) from G6P, in an NAD-dependent aldol cyclization. Its key role in myo-inositol metabolism makes it a putative target for the treatment of several health disorders. The design of these inhibitors allowed for the possibility of substrate-like behavior, reversible inhibition, or mechanism-based inactivation. In this chapter, the synthesis of these compounds, expression and purification of recombinant hexahistidine-tagged mIPS, the mIPS kinetic assay and methods for determining the behavior of the phosphate analogs in the presence of mIPS, and a docking approach to rationalizing the observed behavior are described., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Phosphonates are produced across all domains of life and used widely in medicine and agriculture. Biosynthesis almost universally originates from the enzyme phosphoenolpyruvate mutase (Ppm), EC 5.4.2.9, which catalyzes O-P bond cleavage in phosphoenolpyruvate (PEP) and forms a high energy C-P bond in phosphonopyruvate (PnPy). Mechanistic scrutiny of this unusual intramolecular O-to-C phosphoryl transfer began with the discovery of Ppm in 1988 and concluded in 2008 with computational evidence supporting a concerted phosphoryl transfer via a dissociative metaphosphate-like transition state. This mechanism deviates from the standard 'in-line attack' paradigm for enzymatic phosphoryl transfer that typically involves a phosphoryl-enzyme intermediate, but definitive evidence is sparse. Here we review the experimental evidence leading to our current mechanistic understanding and highlight the roles of previously underappreciated conserved active site residues. We then identify remaining opportunities to evaluate overlooked residues and unexamined substrates/inhibitors., (© 2022 Wiley-VCH GmbH.)

The biosynthesis of myo -inositol (mI) is central to the function of many organisms across all kingdoms of life. The first and rate-limiting step in this pathway is catalyzed by 1l- myo -inositol 1-phosphate synthase (mIPS), which converts d-glucose 6-phosphate (G6P) into 1l- myo -inositol 1-phosphate (mI1P). Extensive studies have shown that this reaction occurs through a stepwise NAD + -dependent redox aldol cyclization mechanism producing enantiomerically pure mI1P. Although the stereochemical nature of the mechanism has been elucidated, there is a lack of understanding of the importance of amino acid residues in the active site. Crystal structures of mIPS in the ternary complex with substrate analogues and NAD(H) show different ligand orientations. We therefore proposed to use isosteric and isoelectronic analogues of G6P to probe the active site. Here, we report the synthesis of the methylenephosphonate, difluoromethylenephosphonate, and ( R )- and ( S )-monofluoromethylenephosphonate analogues of G6P and their evaluation as inhibitors of mIPS activity. While the CH 2 and CF 2 analogues were produced with slight modification of a previously described route, the CHF analogues were synthesized through a new, shorter pathway. Kinetic behavior shows that all compounds are reversible competitive inhibitors with respect to G6P, with K i values in the order CF 2 (0.18 mM) < ( S )-CHF (0.24 mM) < ( R )-CHF (0.59 mM) < CH 2 (1.2 mM). Docking studies of these phosphonates using published crystal structures show that substitution of the oxygen atom of the substrate changes the conformation of the resulting inhibitors, altering the position of carbon-6 and carbon-5, and this change is more pronounced with fluorine substitution.

Gardnerella spp. in the vaginal microbiome are associated with bacterial vaginosis, in which a lactobacillus-dominated community is replaced with mixed bacteria, including Gardnerella species. Co-occurrence of multiple Gardnerella species in the vaginal environment is common, but different species are dominant in different women. Competition for nutrients, including glycogen, could play an important role in determining the microbial community structure. Digestion of glycogen into products that can be taken up and further processed by bacteria requires the combined activities of several enzymes collectively known as amylases, which belong to glycoside hydrolase family 13 (GH13) within the CAZy classification system. GH13 is a large and diverse family of proteins, making prediction of their activities challenging. SACCHARIS annotation of the GH13 family in Gardnerella resulted in identification of protein domains belonging to eight subfamilies. Phylogenetic analysis of predicted amylase sequences from 26 genomes demonstrated that a putative α-glucosidase-encoding sequence, CG400&#95;06090, was conserved in all Gardnerella spp. The predicted α-glucosidase enzyme was expressed, purified, and functionally characterized. The enzyme was active on a variety of maltooligosaccharides with maximum activity at pH 7. K m , k cat , and k cat /K m values for the substrate 4-nitrophenyl α-d-glucopyranoside were 8.3 μM, 0.96 min -1 , and 0.11 μM -1  min -1 , respectively. Glucose was released from maltose, maltotriose, maltotetraose, and maltopentaose, but no products were detected when the enzyme was incubated with glycogen. Our findings show that Gardnerella spp. produce an α-glucosidase enzyme that may contribute to the multistep process of glycogen metabolism by releasing glucose from maltooligosaccharides. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major carbon source available to vaginal bacteria. The significance of our research is characterizing the activity of an enzyme conserved in Gardnerella species that likely contributes to the ability of these bacteria to utilize glycogen.

Kanosamine is an antibiotic and antifungal compound synthesized from glucose 6-phosphate (G6P) in Bacillus subtilis by the action of three enzymes: NtdC, which catalyzes NAD-dependent oxidation of the C3-hydroxyl; NtdA, a PLP-dependent aminotransferase; and NtdB, a phosphatase. We previously demonstrated that NtdC can also oxidize substrates such as glucose and xylose, though at much lower rates, suggesting that the phosphoryloxymethylene moiety of the substrate is critical for effective catalysis. To probe this, we synthesized two phosphonate analogues of G6P in which the bridging oxygen is replaced by methylene and difluoromethylene groups. These analogues are substrates for NtdC, with second-order rate constants an order of magnitude lower than those for G6P. NtdA converts the resulting 3-keto products to the corresponding kanosamine 6-phosphonate analogues. We compared the rates to the rate of NtdC oxidation of glucose and xylose and showed that the low reactivity of xylose could be rescued 4-fold by the presence of phosphite, mimicking G6P in two pieces. These results allow the evaluation of the individual energetic contributions to catalysis of the bridging oxygen, the bridging C6 methylene, the phosphodianion, and the entropic gain of one substrate versus two substrate pieces. Phosphite also rescued the reversible formation 3-amino-3-deoxy-d-xylose by NtdA, demonstrating that truncated and nonhydrolyzable analogues of kanosamine 6-phosphate can be generated enzymatically.

Kanosamine is an antibiotic and antifungal monosaccharide. The kanosamine biosynthetic pathway from glucose 6-phosphate in Bacillus cereus UW85 was recently reported, and the functions of each of the three enzymes in the pathway, KabA, KabB and KabC, were demonstrated. KabA, a member of a subclass of the VI β family of PLP-dependent aminotransferases, catalyzes the second step in the pathway, generating kanosamine 6-phosphate (K6P) using l-glutamate as the amino-donor. KabA catalysis was shown to be extremely efficient, with a second-order rate constant with respect to K6P transamination of over 10 7 M -1 s -1 . Here we report the high-resolution structure of KabA in both the PLP- and PMP-bound forms. In addition, co-crystallization with K6P allowed the structure of KabA in complex with the covalent PLP-K6P adduct to be solved. Co-crystallization or soaking with glutamate or 2-oxoglutarate did not result in crystals with either substrate/product. Reduction of the PLP-KabA complex with sodium cyanoborohydride gave an inactivated enzyme, and crystals of the reduced KabA were soaked with the l-glutamate analog glutarate to mimic the KabA-PLP-l-glutamate complex. Together these four structures give a complete picture of how the active site of KabA recognizes substrates for each half-reaction. The KabA structure is discussed in the context of homologous aminotransferases., (Copyright © 2021 Elsevier Inc. All rights reserved.)

NtdC is an NAD-dependent dehydrogenase that catalyzes the conversion of glucose 6-phosphate (G6P) to 3-oxo-glucose 6-phosphate (3oG6P), the first step in kanosamine biosynthesis in Bacillus subtilis and other closely-related bacteria. The NtdC-catalyzed reaction is unusual because 3oG6P undergoes rapid ring opening, resulting in a 1,3-dicarbonyl compound that is inherently unstable due to enolate formation. We have reported the steady-state kinetic behavior of NtdC, but many questions remain about the nature of this reaction, including whether it is the α-anomer, β-anomer, or open-chain form that is the substrate for the enzyme. Here, we report the synthesis of carbocyclic G6P analogues by two routes, one based upon the Ferrier II rearrangement to generate the carbocycle and one based upon a Claisen rearrangement. We were able to synthesize both pseudo-anomers of carbaglucose 6-phosphate (C6P) using the Ferrier approach, and activity assays revealed that the pseudo-α-anomer is a good substrate for NtdC, while the pseudo-β-anomer and the open-chain analogue, sorbitol 6-phosphate (S6P), are not substrates. A more efficient synthesis of α-C6P was achieved using the Claisen rearrangement approach, which allowed for a thorough evaluation of the NtdC-catalyzed oxidation of α-C6P. The requirement for the α-anomer indicates that NtdC and NtdA, the subsequent enzyme in the pathway, have co-evolved to recognize the α-anomer in order to avoid mutarotation between enzymatic steps.

myo -Inositol (mI) is widely distributed in all domains of life and is important for several cellular functions, including bacterial survival. The enzymes responsible for the bacterial catabolism of mI, encoded in the iol operon, can vary from one organism to another, and these pathways have yet to be fully characterized. We previously identified a new scyllo -inositol dehydrogenase (sIDH) in the iol operon of Lactobacillus casei that can oxidize mI in addition to the natural substrate, scyllo -inositol, but the product of mI oxidation was not determined. Here we report the identification of these metabolites by monitoring the reaction with 13 C nuclear magnetic resonance. We prepared all six singly 13 C-labeled mI isotopomers through a biocatalytic approach and used these labeled inositols as substrates for sIDH. The use of all six singly labeled mI isotopomers allowed for metabolite characterization without isolation steps. sIDH oxidation of mI produces 1l-5- myo -inosose preferentially, but also two minor products, 1d- chiro -inosose and 1l- chiro -inosose. Together with previous crystal structure data for sIDH, we were able to rationalize the observed oxidation preference. Our relatively simple procedure for the preparation of isotopically labeled mI standards can have broad applications for the study of mI biotransformations.

Dihydrodipicolinate synthase (DHDPS) from Campylobacter jejuni is a natively homotetrameric enzyme that catalyzes the first unique reaction of (S)-lysine biosynthesis and is feedback-regulated by lysine through binding to an allosteric site. High-resolution structures of the DHDPS-lysine complex have revealed significant insights into the binding events. One key asparagine residue, N84, makes hydrogen bonds with both the carboxyl and the α-amino group of the bound lysine. We generated two mutants, N84A and N84D, to study the effects of these changes on the allosteric site properties. However, under normal assay conditions, N84A displayed notably lower catalytic activity, and N84D showed no activity. Here we show that these mutations disrupt the quaternary structure of DHDPS in a concentration-dependent fashion, as demonstrated by size-exclusion chromatography, multi-angle light scattering, dynamic light scattering, small-angle X-ray scattering (SAXS) and high-resolution protein crystallography., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Kanosamine is an aminosugar antibiotic, and component of complex antibiotics such as kanamycin. The biosynthesis of kanosamine varies among different bacteria; best known is a pathway starting from UDP-glucose, but Bacillus subtilis can produce kanosamine in a three-step pathway from glucose 6-phosphate. A set of genes proposed to encode a kanosamine pathway has previously been identified within the zwittermicin A gene cluster of Bacillus cereus UW85. These genes, designated kabABC, are similar to the B. subtilis kanosamine pathway genes (ntdABC), but have never been studied experimentally. We have expressed each of the kab genes, and studied the in vitro substrate scope and reaction rates and kinetic mechanisms of all three enzymes. The kab genes encode enzymes that catalyze a route similar to that found in B. subtilis from glucose 6-phosphate to kanosamine, passing through an unusual and unstable 3-keto intermediate. Kinetic studies show the first step in the pathway, the KabC-catalyzed oxidation of glucose 6-phosphate at carbon-3, is very slow relative to the subsequent KabA-catalyzed aminotransferase and KabB-catalyzed phosphatase reactions. KabC differs from its homolog, NtdC, in that it is NADP- rather than NAD-dependent. The KabA kinetic study is the first such report for a kanosamine 6-phosphate aminotransferase, revealing an extremely efficient PLP-dependent reaction. These results show that this kanosamine biosynthesis pathway occurs in more than one organism, and that the reactions are tuned in order to avoid any accumulation of the unstable intermediate., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Many bacteria can use myo-inositol as the sole carbon source using enzymes encoded in the iol operon. The first step is catalyzed by the well-characterized myo-inositol dehydrogenase (mIDH), which oxidizes the axial hydroxyl group of the substrate to form scyllo-inosose. Some bacteria, including Lactobacillus casei, contain more than one apparent mIDH-encoding gene in the iol operon, but such redundant enzymes have not been investigated. scyllo-Inositol, a stereoisomer of myo-inositol, is not a substrate for mIDH, but scyllo-inositol dehydrogenase (sIDH) enzymes have been reported, though never observed to be encoded within the iol operon. Sequences indicate these enzymes are related, but the structural basis by which they distinguish their substrates has not been determined. Here we report the substrate selectivity, kinetics, and high-resolution crystal structures of the proteins encoded by iolG1 and iolG2 from L. casei BL23, which we show encode a mIDH and sIDH, respectively. Comparison of the ternary complex of each enzyme with its preferred substrate reveals the key variations allowing for oxidation of an equatorial versus an axial hydroxyl group. Despite the overall similarity of the active site residues, scyllo-inositol is bound in an inverted, tilted orientation by sIDH relative to the orientation of myo-inositol by mIDH., (Copyright © 2018 Elsevier B.V. All rights reserved.)

NtdA from Bacillus subtilis is a sugar aminotransferase that catalyzes the pyridoxal phosphate-dependent equatorial transamination of 3-oxo-α-D-glucose 6-phosphate to form α-D-kanosamine 6-phosphate. The crystal structure of NtdA shows that NtdA shares the common aspartate aminotransferase fold (Type 1) with residues from both monomers forming the active site. The crystal structures of NtdA alone, co-crystallized with the product α-D-kanosamine 6-phosphate, and incubated with the amine donor glutamate reveal three key structures in the mechanistic pathway of NtdA. The structure of NtdA alone reveals the internal aldimine form of NtdA with the cofactor pyridoxal phosphate covalently attached to Lys-247. The addition of glutamate results in formation of pyridoxamine phosphate. Co-crystallization with kanosamine 6-phosphate results in the formation of the external aldimine. Only α-D-kanosamine 6-phosphate is observed in the active site of NtdA, not the β-anomer. A comparison of the structure and sequence of NtdA with other sugar aminotransferases enables us to propose that the VIβ family of aminotransferases should be divided into subfamilies based on the catalytic lysine motif.