Somchart Maenpuen

Serine hydroxymethyltransferase from Plasmodium vivax is different in substrate specificity from its homologues

The putative gene of Plasmodium vivax serine hydroxymethyltransferase (PvSHMT; EC 2.1.2.1) was cloned and expressed in Escherichia coli. The purified enzyme was shown to be a dimeric protein with a monomeric molecular mass of 49 kDa. PvSHMT has a maximum absorption peak at 422 nm with a molar absorption coefficient of 6370 m−1·cm−1. The Kd for binding of the enzyme and pyridoxal‐5‐phosphate was 0.14 ± 0.01 μm. An alternative assay for measuring the tetrahydrofolate‐dependent SHMT activity based on the coupled reaction with 5,10‐methylenetetrahydrofolate reductase (EC 1.5.1.20) from E. coli was developed. PvSHMT uses a ternary‐complex mechanism with a kcat value of 0.98 ± 0.06 s−1 and Km values of 0.18 ± 0.03 and 0.14 ± 0.02  mm for l‐serine and tetrahydrofolate, respectively. The optimum pH of the SHMT reaction was 8.0 and an Arrhenius’s plot showed a transition temperature of 19 °C. Besides l‐serine, PvSHMT forms an external aldimine complex with d‐serine, l‐alanine, l‐threonine and glycine. PvSHMT also catalyzes the tetrahydrofolate‐independent retro‐aldol cleavage of 3‐hydroxy amino acids. Although l‐serine is a physiological substrate for SHMT in the tetrahydrofolate‐dependent reaction, PvSHMT can also use d‐serine. In the absence of tetrahydrofolate at high pH, PvSHMT forms an enzyme–quinonoid complex with d‐serine, but not with l‐serine, whereas SHMT from rabbit liver was reported to form an enzyme–quinonoid complex with l‐serine. The substrate specificity difference between PvSHMT and the mammalian enzyme indicates the dissimilarity between their active sites, which could be exploited for the development of specific inhibitors against PvSHMT.

Structure and proposed mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae

The formation of H2O2 by the FAD‐dependent l‐α‐glycerophosphate oxidase (GlpO) is important for the pathogenesis of Streptococcus pneumoniae and Mycoplasma pneumoniae. The structurally known GlpO from Streptococcus sp. (SspGlpO) is similar to the pneumococcal protein (SpGlpO) and provides a guide for drug design against that target. However, M. pneumoniae GlpO (MpGlpO), having < 20% sequence identity with structurally known GlpOs, appears to represent a second type of GlpO that we designate as type II GlpOs. In the present study, the recombinant His‐tagged MpGlpO structure is described at an approximate resolution of 2.5 Å, solved by molecular replacement using, as a search model, the Bordetella pertussis protein 3253 (Bp3253), comprising a protein of unknown function solved by structural genomics efforts. Recombinant MpGlpO is an active oxidase with a turnover number of approximately 580 min−1, whereas Bp3253 showed no GlpO activity. No substantial differences exist between the oxidized and dithionite‐reduced MpGlpO structures. Although, no liganded structures were determined, a comparison with the tartrate‐bound Bp3253 structure and consideration of residue conservation patterns guided the construction of a model for l‐α‐glycerophosphate (Glp) recognition and turnover by MpGlpO. The predicted binding mode also appears relevant for the type I GlpOs (such as SspGlpO) despite differences in substrate recognition residues, and it implicates a histidine conserved in type I and II Glp oxidases and dehydrogenases as the catalytic acid/base. The present study provides a solid foundation for guiding further studies of the mitochondrial Glp dehydrogenases, as well as for continued studies of M. pneumoniae and S. pneumoniae glycerol metabolism and the development of novel therapeutics targeting MpGlpO and SpGlpO.

Structures of Plasmodium vivax serine hydroxymethyltransferase: implications for ligand-binding specificity and functional control

Crystal structures of P. vivax serine hydroxymethyltransferase (PvSHMT) in complex with l-serine and with d-serine and 5-formyltetrahydrofolate provide better understanding of ligand binding and the catalytic mechanism. Features that are important for controlling the activity and specificity of PvSHMT such as stereoselectivity and redox status are addressed.

Distinct biochemical properties of human serine hydroxymethyltransferase compared with the Plasmodium enzyme: implications for selective inhibition

Serine hydroxymethyltransferase (SHMT) catalyzes the transfer of a hydroxymethyl group from l‐serine to tetrahydrofolate to yield glycine and 5,10‐methylenetetrahydrofolate. Our previous investigations have shown that SHMTs from Plasmodium spp. (P. falciparum, Pf; P. vivax, Pv) are different from the enzyme from rabbit liver in that Plasmodium SHMT can use d‐serine as a substrate. In this report, the biochemical and biophysical properties of the Plasmodium and the human cytosolic form (hcSHMT) enzymes including ligand binding and kinetics were investigated. The data indicate that, similar to Plasmodium enzymes, hcSHMT can use d‐serine as a substrate. However, hcSHMT displays many properties that are different from those of the Plasmodium enzymes. The molar absorption coefficient of hcSHMT‐bound pyridoxal‐5′‐phosphate (PLP) is much greater than PvSHMT‐bound or PfSHMT‐bound PLP. The binding interactions of hcSHMT and Plasmodium SHMT with d‐serine are different, as only the Plasmodium enzyme undergoes formation of a quinonoid‐like species upon binding to d‐serine. Furthermore, it has been noted that hcSHMT displays strong substrate inhibition by tetrahydrofolate (THF) (at THF > 40 μm), compared with SHMTs from Plasmodium and other species. The pH–activity profile of hcSHMT shows higher activities at lower pH values corresponding to a pKa value of 7.8 ± 0.1. Thiosemicarbazide reacts with hcSHMT following a one‐step model [k1 of 12 ± 0.6 m−1·s−1 and k−1 of (1.0 ± 0.6) × 10−3 s−1], while the same reaction with PfSHMT involves at least three steps. All data indicated that the ligand binding environment of SHMT from human and Plasmodium are different, indicating that it should be possible to develop species‐selective inhibitors in future studies.

Kinetic mechanism of L-α-glycerophosphate oxidase from Mycoplasma pneumoniae.

l–α‐glycerophosphate oxidase is an FAD‐dependent enzyme that catalyzes the oxidation of l–α‐glycerophosphate (Glp) by molecular oxygen to generate dihydroxyacetone phosphate (DHAP) and hydrogen peroxide (H2O2). The catalytic properties of recombinant His6‐GlpO from Mycoplasma pneumoniae (His6‐MpGlpO) were investigated through transient and steady‐state kinetics and ligand binding studies. The results indicate that the reaction mechanism of His6‐MpGlpO follows a ping‐pong model. Double‐mixing mode stopped‐flow experiments show that, after flavin‐mediated substrate oxidation, DHAP leaves rapidly prior to the oxygen reaction. The values determined for the individual rate constants and kcat (4.2 s−1 at 4 °C), in addition to the finding that H2O2 binds to the oxidized enzyme, suggest that H2O2 release is the rate‐limiting step for the overall reaction. The results indicate that His6‐MpGlpO contains mixed populations of fast‐ and slow‐reacting species. It is predominantly the fast‐reacting species that participates in turnover. In contrast to other GlpO enzymes previously described, His6‐MpGlpO is able to catalyze the reverse reaction of reduced enzyme and DHAP. This result may be explained by the standard reduction potential value of His6‐MpGlpO (−167 ± 1 mV), which is lower than those of GlpO from other species. We found that d,l–glyceraldehyde 3–phosphate (GAP) may be used as a substrate in the His6‐MpGlpO reaction, although it exhibited an approximately 100‐fold lower kcat value in comparison with the reaction of Glp. These results also imply involvement of GlpO in glycolysis, as well as in lipid and glycerol metabolism. The kinetic models and distinctive properties of His6‐MpGlpO reported here should be useful for future drug development against Mycoplasma pneumoniae infection.

Biotransformation of Plant-Derived Phenolic Acids

Phenolic acids are abundant biomass feedstock that can be derived from the processing of lignin or other byproducts from agro‐industrial waste. Although phenolic acids such as p‐hydroxybenzoic acid, p‐coumaric acid, caffeic acid, vanillic acid, cinnamic acid, gallic acid, syringic acid, and ferulic acid can be used directly in various applications, their value can be significantly increased when they are further modified to high value‐added compounds. This review summarizes and discusses the new advances in cell‐free and whole‐cell biocatalysis technologies for reactions important for conversion of phenolic acids including esterification, decarboxylation, amination, halogenation, hydroxylation, and ring‐breakage reactions. The products of these reactions are useful for the pharmaceutical, cosmetic, food, fragrance, and polymer industries. Production of phenolic acids is sustainable, and these processes for their biotransformation are clean technologies that do not produce toxic waste and use less energy than conventional physical and chemical methods. Thus, biotransformation of phenolic acids provides an economically viable and sustainable means for producing useful materials for society.

Kinetic Mechanism and the Rate-limiting Step of Plasmodium vivax Serine Hydroxymethyltransferase

Background: Plasmodium vivax serine hydroxymethyltransferase (PvSHMT) catalyzes formation of glycine from l-serine and tetrahydrofolate. Results: Results indicate that PvSHMT can bind to either substrate first. The rate constant of glycine formation is similar to kcat. Conclusion: PvSHMT reaction occurs via a random-order mechanism and glycine formation is the rate-limiting step. Significance: The data are useful for future investigation on inhibition of SHMT for antimalarial drug development. Serine hydroxymethyltransferase (SHMT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyzes a hydroxymethyl group transfer from l-serine to tetrahydrofolate (H4folate) to yield glycine and 5,10-methylenetetrahydrofolate (CH2-H4folate). SHMT is crucial for deoxythymidylate biosynthesis and a target for antimalarial drug development. Our previous studies indicate that PvSHMT catalyzes the reaction via a ternary complex mechanism. To define the kinetic mechanism of this catalysis, we explored the PvSHMT reaction by employing various methodologies including ligand binding, transient, and steady-state kinetics as well as product analysis by rapid-quench and HPLC/MS techniques. The results indicate that PvSHMT can bind first to either l-serine or H4folate. The dissociation constants for the enzyme·l-serine and enzyme·H4folate complexes were determined as 0.18 ± 0.08 and 0.35 ± 0.06 mm, respectively. The amounts of glycine formed after single turnovers of different preformed binary complexes were similar, indicating that the reaction proceeds via a random-order binding mechanism. In addition, the rate constant of glycine formation measured by rapid-quench and HPLC/MS analysis is similar to the kcat value (1.09 ± 0.05 s−1) obtained from the steady-state kinetics, indicating that glycine formation is the rate-limiting step of SHMT catalysis. This information will serve as a basis for future investigation on species-specific inhibition of SHMT for antimalarial drug development.

Human and Plasmodium serine hydroxymethyltransferases differ in rate-limiting steps and pH-dependent substrate inhibition behavior

Serine hydroxymethyltransferase (SHMT), an essential enzyme for cell growth and development, catalyzes the transfer of -CH2OH from l-serine to tetrahydrofolate (THF) to form glycine and 5,10-methylenetetrahydrofolate (MTHF) which is used for nucleotide synthesis. Insights into the ligand binding and inhibition properties of human cytosolic SHMT (hcSHMT) and Plasmodium SHMT (PvSHMT) are crucial for designing specific drugs against malaria and cancer. The results presented here revealed strong and pH-dependent THF inhibition of hcSHMT. In contrast, in PvSHMT, THF inhibition and the influence of pH were not as pronounced. Ligand binding experiments performed at various pH values indicated that the hcSHMT:Gly complex binds THF more tightly at lower pH conditions, while the binding affinity of the PvSHMT:Gly complex for THF is not pH-dependent. Pre-steady state kinetic (rapid-quench) analysis of hcSHMT showed burst kinetics, indicating that glycine formation occurs fastest in the first turnover relative to the subsequent turnovers i.e. glycine release is the rate-limiting step in the hcSHMT reaction. All data suggest that excess THF likely binds E:Gly binary complex and forms the E:Gly:THF dead-end complex before glycine is released. A unique flap motif found in the structure of hcSHMT may be the key structural feature that imparts these described characteristics of hcSHMT.

3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Pseudomonas aeruginosa: An Fe(II)-containing enzyme with fast turnover.

3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from Pseudomonas aeruginosa (PaDHPAO) was overexpressed in Escherichia coli and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM-1cm-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a Km for DHPA of 58 ± 8 μM and a kcat of 64 s-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(k’cat) versus the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (Ea) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology.

Selective determination of the catalytic cysteine pKa of two‐cysteine succinic semialdehyde dehydrogenase from Acinetobacter baumannii using burst kinetics and enzyme adduct formation

Succinic semialdehyde dehydrogenase (SSADH) from Acinetobacter baumannii (Ab) catalyzes the oxidation of succinic semialdehyde (SSA). This enzyme has two conserved cysteines (Cys289 and Cys291) and preferentially uses NADP+ over NAD+ as a hydride acceptor. Steady‐state kinetic analysis showed that AbSSADH has the highest catalytic turnover (137 s−1) and can tolerate SSA inhibition the most (< 500 μm) among all SSADHs reported. Alanine substitutions of the two conserved cysteines indicated that Cys291Ala has ~ 65% activity compared with the wild‐type enzyme while Cys289Ala is inactive, suggesting that Cys289 is the active residue participating in catalysis. Pre‐steady‐state kinetics showed for the first time burst kinetics for NADPH formation in SSADH, indicating that the rate‐limiting step is associated with steps that occur after the hydride transfer. As the magnitude of burst kinetics represents the amount of NADPH formed during the first turnover, it is directly dependent on the amount of the deprotonated form of cysteine. The pKa of Cys289 was calculated from a plot of the burst magnitude vs pH as 7.4 ± 0.2. The Cys289 pKa was also measured based on the ability of AbSSADH to form an NADP–cysteine adduct, which can be detected by the increase of absorbance at ~ 330 nm as 7.9 ± 0.2. The lowering of the catalytic cysteine pKa by 0.6–1 unit renders the catalytic thiol more nucleophilic, which facilitates AbSSADH catalysis under physiological conditions. The methods established herein can specifically measure the active site cysteine pKa without interference from other cysteines. These techniques may be useful for studying ionization state of other cysteine‐containing aldehyde dehydrogenases.