CMP-N-Acetylneuraminic Acid Synthetase from Escherichia coli K1 Is a Bifunctional Enzyme

Abstract

Escherichia coli CMP-NeuAc synthetase (EC 2.7.7.43) catalyzes the synthesis of CMP-NeuAc from CTP and NeuAc, which is essential for the formation of capsule polysialylate for strain K1. Alignment of the amino acid sequence of E. coli CMP-NeuAc synthetase with those from other bacterial species revealed that the conserved motifs were located in its N termini, whereas the C terminus appeared to be redundant. Based on this information, a series of deletions from the 3-end of the CMPNeuAc synthetase coding region was constructed and expressed in E. coli. As a result, the catalytic domain required for CMP-NeuAc synthetase was found to be in the N-terminal half consisting of amino acids 1-229. Using the strategy of tertiary structure prediction based on the homologous search of the secondary structure, the C-terminal half was recognized as an ␣1-subunit of bovine brain platelet-activating factor acetylhydrolase isoform I. The biochemical analyses showed that the C-terminal half consisting of amino acids 228 -418 exhibited platelet-activating factor acetylhydrolase activity. The enzyme properties and substrate specificity were similar to that of bovine brain ␣1-subunit. Although its physiological function is still unclear, it has been proposed that the ␣1-subunit-like domain of E. coli may be involved in the traversal of the blood-brain barrier.

Sialic Acid Metabolism and Systemic Pasteurellosis

“…f H. influenzae strain Rd (17). g pm1710 also encodes a region that is 60% identical over 198 amino acid residues and 57% identical over 195 residues with the putative lysophospholipases of Actinobacillus pleuropneumoniae serovar 1 strain 4074 and H. somnus strain 2336, respectively, a finding consistent with a report that E. coli NeuA is a bifunctional enzyme (31).…”

Serum Activity of Platelet-Activating Factor Acetylhydrolase Is a Potential Clinical Marker for Leptospirosis Pulmonary Hemorrhage

“…Although, so far, there is no complete data available for enzyme kinetic parameters of the serum PAF-AH towards this artificial substrate, its Km was determined in this study, which is similar to that of the L-PAF-AH. Therefore, in contrast to the analogous activity found in a bifunctional CMP- N -acetylneuraminic acid synthetase (CS) encoded by a pathogenic E. coli K1 as one of its two catalytic domains [22], this L-PAF-AH is an independent single-chain enzyme, with its structure similar to that of the mammalian isoform I α1-subunit while its substrate specificity being different from that of the E. coli CS but similar to the serum counterpart enzyme.…”

“…The PAF-AH domain of the E. coli CS is highly similar to the α1-subunit of PAF-AH isoform I with respect to both its amino acid sequence and the computer simulated 3-D structure. Thus, it is no surprise that the PAF-AH domain of the E. coli CS exhibited similar substrate specificity as that of the α1-subunit [22]. The amino acid residues corresponding to Leu 48 , Leu 194 and Thr 103 of the α1-subunit were also conserved in the E. coli CS (identified as Leu 258 , Leu 399 and Thr 308 based on three-dimensional modeling) [22].…”

Exploring the Potent Inhibition of CTP Synthase by Gemcitabine‐5′‐Triphosphate

“…The glutamine amidotransferase cytidine-5'-triphosphate synthase [EC 6.3.4.2;U TP:ammonia ligase (ADP-forming);C TPS] catalyzes the ATP-dependentf ormation of CTP from UTP using either l-glutamine (Gln) or NH 3 as the nitrogen source (Scheme 1). [1] Because this is the sole route for the de novo biosynthesis of the cytosine base, and also in view of the central role of CTP in the biosynthesis of nucleic acids, [2] phospholipids via the Kennedy pathway, [3,4] and sialic acid, [5] CTPS is an attractive target for the development of antiviral, [6,7] antiprotozoal, [8][9][10][11][12][13][14] and antineoplastic agents. [2,15,16] CTPS is also ar ecognized target for immunosuppressivet herapy due to its importance in T-cell proliferation.…”