Conformational change of organic cofactor PLP is essential for catalysis in PLP-dependent enzymes.

Pyridoxal 5'-phosphate (PLP)-dependent enzymes are ubiquitous, catalyzing various biochemical reactions of approximately 4% of all classified enzymatic activities. They transform amines and amino acids into important metabolites or signaling molecules and are important drug targets in many diseases. In the crystal structures of PLP-dependent enzymes, organic cofactor PLP showed diverse conformations depending on the catalytic step. The conformational change of PLP is essential in the catalytic mechanism. In the study, we review the sophisticated catalytic mechanism of PLP, especially in transaldimination reactions. Most drugs targeting PLP-dependent enzymes make a covalent bond to PLP with the transaldimination reaction. A detailed understanding of organic cofactor PLP will help develop a new drug against PLP-dependent enzymes. [BMB Reports 2022; 55(9): 439-446].

Crystal structures of an atypical aldehyde dehydrogenase having bidirectional oxidizing and reducing activities.

Aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent oxidoreductases that catalyze the oxidation of a variety of aldehydes to their acid forms. In this study, we determined the crystal structures of ALDH from Bacillus cereus (BcALDH), alone, and in complex with NAD+ and NADP+. This enzyme can oxidize all-trans-retinal to all-trans-retinoic acid using either NAD+ or NADP+ with equal efficiency, and atypically, as a minor activity, can reduce all-trans-retinal to all-trans-retinol using NADPH. BcALDH accommodated the additional 2'-phosphate of NADP+ by expanding the cofactor-binding pocket and upshifting the AMP moiety in NADP+. The nicotinamide moiety in NAD+ and NADP+ had direct interactions with the conserved catalytic residues (Cys300 and Glu266) and caused concerted conformational changes. We superimposed the structure of retinoic acid bound to human ALDH1A3 onto the BcALDH structure and speculated a model of the substrate all-trans-retinal bound to BcALDH. We also proposed a plausible mechanism for the minor reducing activity of BcALDH. These BcALDH structures will be useful in understanding cofactor specificity and the catalytic mechanism of an atypical bacterial BcALDH and should help the development of a new biocatalyst to produce retinoic acid and related high-end products.

Crystal Structures of Peptide Deformylase from Rice Pathogen Xanthomonas oryzae pv. oryzae in Complex with Substrate Peptides, Actinonin, and Fragment Chemical Compounds.

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight on rice; this species is one of the most destructive pathogenic bacteria in rice cultivation worldwide. Peptide deformylase (PDF) catalyzes the removal of the N-formyl group from the N-terminus of newly synthesized polypeptides in bacterial cells and is an important target to develop antibacterial agents. We determined crystal structures of Xoo PDF (XoPDF) at up to 1.9 Å resolution, which include apo, two substrate-bound (methionine-alanine or methionine-alanine-serine), an inhibitor-bound (actinonin), and six fragment chemical-bound structures. Six fragment chemical compounds were bound in the substrate-binding pocket. The fragment chemical-bound structures were compared to the natural PDF inhibitor actinonin-bound structure. The fragment chemical molecules will be useful to design an inhibitor specific to XoPDF and a potential pesticide against Xoo.

Alternative Biotransformation of Retinal to Retinoic Acid or Retinol by an Aldehyde Dehydrogenase from Bacillus cereus.

UNLABELLED: A novel bacterial aldehyde dehydrogenase (ALDH) that converts retinal to retinoic acid was first identified in Bacillus cereus The amino acid sequence of ALDH from B. cereus (BcALDH) was more closely related to mammalian ALDHs than to bacterial ALDHs. This enzyme converted not only small aldehydes to carboxylic acids but also the large aldehyde all-trans-retinal to all-trans-retinoic acid with NAD(P)(+) We newly found that BcALDH and human ALDH (ALDH1A1) could reduce all-trans-retinal to all-trans-retinol with NADPH. The catalytic residues in BcALDH were Glu266 and Cys300, and the cofactor-binding residues were Glu194 and Glu457. The E266A and C300A variants showed no oxidation activity. The E194S and E457V variants showed 15- and 7.5-fold higher catalytic efficiency (kcat/Km) for the reduction of all-trans-retinal than the wild-type enzyme, respectively. The wild-type, E194S variant, and E457V variant enzymes with NAD(+) converted 400 µM all-trans-retinal to 210 µM all-trans-retinoic acid at the same amount for 240 min, while with NADPH, they converted 400 µM all-trans-retinal to 20, 90, and 40 µM all-trans-retinol, respectively. These results indicate that BcALDH and its variants are efficient biocatalysts not only in the conversion of retinal to retinoic acid but also in its conversion to retinol with a cofactor switch and that retinol production can be increased by the variant enzymes. Therefore, BcALDH is a novel bacterial enzyme for the alternative production of retinoic acid and retinol. IMPORTANCE: Although mammalian ALDHs have catalyzed the conversion of retinal to retinoic acid with NAD(P)(+) as a cofactor, a bacterial ALDH involved in the conversion is first characterized. The biotransformation of all-trans-retinal to all-trans-retinoic acid by BcALDH and human ALDH was altered to the biotransformation to all-trans-retinol by a cofactor switch using NADPH. Moreover, the production of all-trans-retinal to all-trans-retinol was changed by mutations at positions 194 and 457 in BcALDH. The alternative biotransformation of retinoids was first performed in the present study. These results will contribute to the biotechnological production of retinoids, including retinoic acid and retinol.

Structure of D-alanine-D-alanine ligase from Yersinia pestis: nucleotide phosphate recognition by the serine loop.

D-Alanyl-D-alanine is an essential precursor of bacterial peptidoglycan and is synthesized by D-alanine-D-alanine ligase (DDL) with hydrolysis of ATP; this reaction makes DDL an important drug target for the development of antibacterial agents. Five crystal structures of DDL from Yersinia pestis (YpDDL) were determined at 1.7-2.5 Å resolution: apo, AMP-bound, ADP-bound, adenosine 5'-(ß,γ-imido)triphosphate-bound, and D-alanyl-D-alanine- and ADP-bound structures. YpDDL consists of three domains, in which four loops, loop 1, loop 2 (the serine loop), loop 3 (the ω-loop) and loop 4, constitute the binding sites for two D-alanine molecules and one ATP molecule. Some of them, especially the serine loop and the ω-loop, show flexible conformations, and the serine loop is mainly responsible for the conformational change in substrate nucleotide phosphates. Enzyme-kinetics assays were carried out for both the D-alanine and ATP substrates and a substrate-binding mechanism was proposed for YpDDL involving conformational changes of the loops.

Characterization of alcohol dehydrogenase from Kangiella koreensis and its application to production of all-trans-retinol.

A recombinant alcohol dehydrogenase (ADH) from Kangiella koreensis was purified as a 40 kDa dimer with a specific activity of 21.3 nmol min(-1) mg(-1), a K m of 1.8 µM, and a k cat of 1.7 min(-1) for all-trans-retinal using NADH as cofactor. The enzyme showed activity for all-trans-retinol using NAD (+) as a cofactor. The reaction conditions for all-trans-retinol production were optimal at pH 6.5 and 60 °C, 2 g enzyme l(-1), and 2,200 mg all-trans-retinal l(-1) in the presence of 5% (v/v) methanol, 1% (w/v) hydroquinone, and 10 mM NADH. Under optimized conditions, the ADH produced 600 mg all-trans-retinol l(-1) after 3 h, with a conversion yield of 27.3% (w/w) and a productivity of 200 mg l(-1) h(-1). This is the first report of the characterization of a bacterial ADH for all-trans-retinal and the biotechnological production of all-trans-retinol using ADH.

Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine ß-lyase from Acinetobacter baumannii OXA-23.

Multidrug-resistant Acinetobacter baumannii (Ab) has emerged as a leading nosocomial pathogen because of its resistance to most currently available antibiotics. Cystathionine ß-lyase (CBL), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, catalyzes the second step in the transsulfuration pathway, which is essential for the metabolic interconversion of the sulfur-containing amino acids homocysteine and methionine. The enzymes of the transsulfuration pathway are considered to be attractive drug targets owing to their specificity to microbes and plants. As a potential target for the development of novel antibacterial drugs, the AbCBL protein was expressed, purified and crystallized. An AbCBL crystal diffracted to 1.57 Šresolution and belonged to the trigonal space group P3112, with unit-cell parameters a = b = 102.9, c = 136.5 Å. The asymmetric unit contained two monomers, with a corresponding VM of 2.3 Å(3) Da(-1) and a solvent content of 46.9%.

Crystallization and preliminary X-ray crystallographic analysis of the XoGroEL chaperonin from Xanthomonas oryzae pv. oryzae.

Along with the co-chaperonin GroES, the chaperonin GroEL plays an essential role in enhancing protein folding or refolding and in protecting proteins against misfolding and aggregation in the cellular environment. The XoGroEL gene (XOO_4288) from Xanthomonas oryzae pv. oryzae was cloned and the protein was expressed, purified and crystallized. The purified XoGroEL protein was crystallized using the hanging-drop vapour-diffusion method and a crystal diffracted to a resolution of 3.4 Å. The crystal belonged to the orthorhombic space group P212121 with 14 monomers in the asymmetric unit, with a corresponding VM of 2.7 Å(3) Da(-1) and a solvent content of 54.5%.

Expression, crystallization and preliminary X-ray crystallographic analysis of DNA-directed RNA polymerase subunit L from Thermococcus onnurineus NA1.

RNA polymerase (RNAP) plays a crucial role in gene expression in all organisms. It is a multiprotein complex that produces primary transcript RNA. Generally, the basal transcription apparatus in archaea is simpler than the eukaryotic RNA polymerase II counterpart. To understand the structure and function of archaeal RNAP, the TON-0309 gene encoding DNA-directed RNA polymerase subunit L (ToRNAP_L) from Thermococcus onnurineus NA1 was cloned and the protein was overexpressed in Escherichia coli, purified and crystallized. The purified protein was crystallized using the hanging-drop vapour-diffusion method and the crystal diffracted to 2.10 Å resolution. The crystal belonged to the hexagonal space group P6122, with unit-cell parameters a = b = 42.3, c = 211.2 Å. One molecule was present in the asymmetric unit, with a corresponding VM of 2.5 Å(3) Da(-1) and a solvent content of 50.0%.

Divalent metal ion-based catalytic mechanism of the Nudix hydrolase Orf153 (YmfB) from Escherichia coli.

YmfB from Escherichia coli is the Nudix hydrolase involved in the metabolism of thiamine pyrophosphate, an important compound in primary metabolism and a cofactor of many enzymes. In addition, it hydrolyzes (d)NTPs to (d)NMPs and inorganic orthophosphates in a stepwise manner. The structures of YmfB alone and in complex with three sulfates and two manganese ions determined by X-ray crystallography, when compared with the structures of other Nudix hydrolases such as MutT, Ap4Aase and DR1025, provide insight into the unique hydrolysis mechanism of YmfB. Mass-spectrometric analysis confirmed that water attacks the terminal phosphates of GTP and GDP sequentially. Kinetic analysis of binding-site mutants showed that no individual residue is absolutely required for catalytic activity, suggesting that protein residues do not participate in the deprotonation of the attacking water. Thermodynamic integration calculations show that a hydroxyl ion bound to two divalent metal ions attacks the phosphate directly without the help of a nearby catalytic base.

Expression, crystallization and preliminary X-ray crystallographic analysis of D-alanine-D-alanine ligase from OXA-23-producing Acinetobacter baumannii K0420859.

Acinetobacter baumannii causes bacteraemia, pneumonia, other respiratory-tract and urinary-tract infections in humans. OXA-23 carbapenemase-producing A. baumannii K0420859 (A. baumannii OXA-23) is resistant to carbapenem, a common antibacterial drug. To develop an efficient and novel antibacterial drug against A. baumannii OXA-23, D-alanine-D-alanine ligase, which is essential in bacterial cell-wall synthesis, is of interest. Here, the D-alanine-D-alanine ligase (AbDdl) gene from A. baumannii OXA-23 was cloned and expressed, and the AbDdl protein was purified and crystallized; this enzyme can be used as a novel target for an antibacterial drug against A. baumannii OXA-23. The AbDdl crystal diffracted to a resolution of 2.8 Šand belonged to the orthorhombic space group P212121, with unit-cell parameters a = 113.4, b = 116.7, c = 176.5 Å, a corresponding VM of 2.8 Å(3) Da(-1) and a solvent content of 56.3%, and six protomers in the asymmetric unit.

PLP undergoes conformational changes during the course of an enzymatic reaction.

Numerous enzymes, such as the pyridoxal 5'-phosphate (PLP)-dependent enzymes, require cofactors for their activities. Using X-ray crystallography, structural snapshots of the L-serine dehydratase catalytic reaction of a bacterial PLP-dependent enzyme were determined. In the structures, the dihedral angle between the pyridine ring and the Schiff-base linkage of PLP varied from 18° to 52°. It is proposed that the organic cofactor PLP directly catalyzes reactions by active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by high-level quantum-mechanical calculations. The conformational change was essential for nucleophilic attack of the substrate on PLP, for concerted proton transfer from the substrate to the protein and for directing carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions, like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying the previously unknown catalytic roles of organic cofactors.

Crystal structures of d-alanine-d-alanine ligase from Xanthomonas oryzae pv. oryzae alone and in complex with nucleotides.

D-Alanine-D-alanine ligase (DDL) catalyzes the biosynthesis of d-alanyl-d-alanine, an essential bacterial peptidoglycan precursor, and is an important drug target for the development of antibacterials. We determined four different crystal structures of DDL from Xanthomonas oryzae pv. oryzae (Xoo) causing Bacteria Blight (BB), which include apo, ADP-bound, ATP-bound, and AMPPNP-bound structures at the resolution between 2.3 and 2.0 Å. Similarly with other DDLs, the active site of XoDDL is formed by three loops from three domains at the center of enzyme. Compared with d-alanyl-d-alanine and ATP-bound TtDDL structure, the γ-phosphate of ATP in XoDDL structure was shifted outside toward solution. We swapped the ω-loop (loop3) of XoDDL with those of Escherichia coli and Helicobacter pylori DDLs, and measured the enzymatic kinetics of wild-type XoDDL and two mutant XoDDLs with the swapped ω-loops. Results showed that the direct interactions between ω-loop and other two loops are essential for the active ATP conformation for D-ala-phosphate formation.

Expression, crystallization and preliminary X-ray crystallographic analysis of peptide deformylase from Campylobacter jejuni.

Campylobacter jejuni is one of the major foodborne pathogens causing human infection. Peptide deformylase, a metallohydrolase, catalyzes the deformylation of N-formylated methionine in newly synthesized polypeptides in prokaryotes and some eukaryotic organelles. The deformylation process is an essential step in protein synthesis and has attracted much attention as a potential target for the development of novel antibacterial agents. Here, the cloned codon-optimized def gene from C. jejuni was synthesized and the protein was expressed, purified and crystallized. C. jejuni peptide deformylase crystals obtained at pH 7.0 and pH 6.5 diffracted to 2.9 Šresolution and belonged to the trigonal space group R3, with unit-cell parameters a=b=105.7, c=58.0 Å. One monomer existed in the asymmetric unit, with a corresponding VM of 3.1 Å3 Da(-1) and a solvent content of 60.4%.

Expression, crystallization and preliminary X-ray crystallographic analysis of cellobiose 2-epimerase from Dictyoglomus turgidum DSM 6724.

Cellobiose 2-epimerase epimerizes and isomerizes ß-1,4- and α-1,4-gluco-oligosaccharides. N-Acyl-D-glucosamine 2-epimerase (DT_epimerase) from Dictyoglomus turgidum has an unusually high catalytic activity towards its substrate cellobiose. DT_epimerase was expressed, purified and crystallized. Crystals were obtained of both His-tagged DT_epimerase and untagged DT_epimerase. The crystals of His-tagged DT_epimerase diffracted to 2.6 Šresolution and belonged to the monoclinic space group P21, with unit-cell parameters a=63.9, b=85.1, c=79.8 Å, ß=110.8°. With a Matthews coefficient VM of 2.18 Å3 Da(-1), two protomers were expected to be present in the asymmetric unit with a solvent content of 43.74%. The crystals of untagged DT_epimerase diffracted to 1.85 Šresolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a=55.9, b=80.0, c=93.7 Å. One protomer in the asymmetric unit was expected, with a corresponding VM of 2.26 Å3 Da(-1) and a solvent content of 45.6%.

Expression, crystallization and preliminary X-ray crystallographic analysis of alanine racemase from Acinetobacter baumannii OXA-23.

Acinetobacter baumannii has received much attention owing to its exceptional ability to develop resistance to currently available antibiotics. Alanine racemase (ALR) catalyzes the racemization of L-alanine to D-alanine with pyridoxal 5'-phosphate (PLP) as a cofactor. The D-alanine product is an essential component of the bacterial cell wall and ALR is a potential target for the development of novel antibacterial drugs. The alr gene from A. baumannii was cloned and the protein (AbALR) was expressed, purified and crystallized. The AbALR crystal diffracted to 2.3 Šresolution and belonged to the primitive orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.1, b = 85.0, c = 167.7 Å. Two protomers were present in the asymmetric unit, with a corresponding V(M) value of 2.3 Å(3) Da(-1) and a solvent content of 47.5%.

Expression, crystallization and preliminary X-ray crystallographic analysis of alcohol dehydrogenase (ADH) from Kangiella koreensis.

Alcohol dehydrogenases (ADHs) are a group of dehydrogenase enzymes that facilitate the interconversion between alcohols and aldehydes or ketones with the reduction of NAD(+) to NADH. In bacteria, some alcohol dehydrogenases catalyze the opposite reaction as part of fermentation to ensure a constant supply of NAD(+). The adh gene from Kangiella koreensis was cloned and the protein (KkADH) was expressed, purified and crystallized. A KkADH crystal diffracted to 2.5 Šresolution and belonged to the monoclinic space group P2(1), with unit-cell parameters a = 94.1, b = 80.9, c = 115.6 Å, ß = 111.9°. Four monomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.

Expression, crystallization and preliminary X-ray crystallographic analysis of aldehyde dehydrogenase (ALDH) from Bacillus cereus.

Aldehyde dehydrogenase (ALDH) catalyses the oxidation of aldehydes using NAD(P)(+) as a cofactor. Most aldehydes are toxic at low levels. ALDHs are used to regulate metabolic intermediate aldehydes. The aldh gene from Bacillus cereus was cloned and the ALDH protein was expressed, purified and crystallized. A crystal of the ALDH protein diffracted to 2.6 Šresolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 83.5, b = 93.3, c = 145.5 Å, ß = 98.05°. Four protomers were present in the asymmetric unit, with a corresponding VM of 2.55 Å(3) Da(-1) and a solvent content of 51.8%.

Expression, crystallization and preliminary X-ray crystallographic analysis of cystathionine γ-synthase (XometB) from Xanthomonas oryzae pv. oryzae.

Cystathionine γ-synthase (CGS) catalyzes the first step in the transsulfuration pathway leading to the formation of cystathionine from O-succinylhomoserine and L-cysteine through a γ-replacement reaction. As an antibacterial drug target against Xanthomonas oryzae pv. oryzae (Xoo), CGS from Xoo (XometB) was cloned, expressed, purified and crystallized. The XometB crystal diffracted to 2.4 Šresolution and belonged to the tetragonal space group I4(1), with unit-cell parameters a=b=165.4, c=241.7 Å. There were four protomers in the asymmetric unit, with a corresponding solvent content of 73.9%.

Crystal structure of malonyl CoA-Acyl carrier protein transacylase from Xanthomanous oryzae pv. oryzae and its proposed binding with ACP.

Xanthomonas oryzae pv. oryzae (Xoo) is a plant bacterial pathogen that causes bacterial blight (BB) disease, resulting in serious production losses of rice. The crystal structure of malonyl CoA-acyl carrier protein transacylase (XoMCAT), encoded by the gene fabD (Xoo0880) from Xoo, was determined at 2.3 Å resolution in complex with N-cyclohexyl-2-aminoethansulfonic acid. Malonyl CoA-acyl carrier protein transacylase transfers malonyl group from malonyl CoA to acyl carrier protein (ACP). The transacylation step is essential in fatty acid synthesis. Based on the rationale, XoMCAT has been considered as a target for antibacterial agents against BB. Protein-protein interaction between XoMCAT and ACP was also extensively investigated using computational docking, and the proposed model revealed that ACP bound to the cleft between two XoMCAT subdomains.