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Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis.

ABSTRACT: N-Acetylneuraminic acid lyase (NAL, E.C. number 4.1.3.3) is a Class I aldolase that catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) from pyruvate and N-acetyl-D-mannosamine (ManNAc). Due to the high Neu5Ac cleavage activity in most isozyme forms, the enzyme catalyzes the rate-limiting step of two biocatalytic reactions producing Neu5Ac in industry. We report the biochemical characterization of a novel NAL from a "GRAS" (General recognized as safe) strain C. glutamicum ATCC 13032 (CgNal). Compared to all previously reported NALs, CgNal exhibited the lowest kcat/Km value for Neu5Ac and highest kcat/Km values for ManNAc and pyruvate, which makes CgNal favor industrial Neu5Ac synthesis process in a non-equilibrium condition. The recombinant CgNal reached the highest expression level (480 mg/L culture), and the highest reported yield of Neu5Ac was achieved (194 g/L, 0.63 M). All these unique properties make CgNal a promising biocatalyst for industrial Neu5Ac biosynthesis. Additionally, although showing the best Neu5Ac synthesis activity among the NAL family, CgNal is more related to dihydrodipicolinate synthase (DHDPS) by phylogenetic analysis. The activities of CgNal towards both NAL's and DHDPS' substrates are fairly high, which indicates CgNal a bi-functional enzyme. The sequence analysis suggests that CgNal might have adopted a unique set of residues for substrates recognition.

SUBMITTER: Ji W

PROVIDER: S-EPMC5380162 | biostudies-literature | 2015 Mar

REPOSITORIES: biostudies-literature

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Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis.

Ji Wenyan W Sun Wujin W Feng Jinmei J Song Tianshun T Zhang Dalu D Ouyang Pingkai P Gu Zhen Z Xie Jingjing J

Scientific reports 20150323

N-Acetylneuraminic acid lyase (NAL, E.C. number 4.1.3.3) is a Class I aldolase that catalyzes the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac) from pyruvate and N-acetyl-D-mannosamine (ManNAc). Due to the high Neu5Ac cleavage activity in most isozyme forms, the enzyme catalyzes the rate-limiting step of two biocatalytic reactions producing Neu5Ac in industry. We report the biochemical characterization of a novel NAL from a "GRAS" (General recognized as safe) strain C. glutamicum ...[more]

PMID: 25799411

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Project description:N-Acetylneuraminic acid lyase (NAL) is a Class I aldolase that catalyzes the reversible condensation of pyruvate with N-acetyl-d-mannosamine (ManNAc) to yield the sialic acid N-acetylneuraminic acid (Neu5Ac). Aldolases are finding increasing use as biocatalysts for the stereospecific synthesis of complex molecules. Incomplete understanding of the mechanism of catalysis in aldolases, however, can hamper development of new enzyme activities and specificities, including control over newly generated stereocenters. In the case of NAL, it is clear that the enzyme catalyzes a Bi-Uni ordered condensation reaction in which pyruvate binds first to the enzyme to form a catalytically important Schiff base. The identity of the residues required for catalysis of the condensation step and the nature of the transition state for this reaction, however, have been a matter of conjecture. In order to address, this we crystallized a Y137A variant of the E. coli NAL in the presence of Neu5Ac. The three-dimensional structure shows a full length sialic acid bound in the active site of subunits A, B, and D, while in subunit C, discontinuous electron density reveals the positions of enzyme-bound pyruvate and ManNAc. These 'snapshot' structures, representative of intermediates in the enzyme catalytic cycle, provided an ideal starting point for QM/MM modeling of the enzymic reaction of carbon-carbon bond formation. This revealed that Tyr137 acts as the proton donor to the aldehyde oxygen of ManNAc during the reaction, the activation barrier is dominated by carbon-carbon bond formation, and proton transfer from Tyr137 is required to obtain a stable Neu5Ac-Lys165 Schiff base complex. The results also suggested that a triad of residues, Tyr137, Ser47, and Tyr110 from a neighboring subunit, are required to correctly position Tyr137 for its function, and this was confirmed by site-directed mutagenesis. This understanding of the mechanism and geometry of the transition states along the C-C bond-forming pathway will allow further development of these enzymes for stereospecific synthesis of new enzyme products.

Project description:Background:N-glycolylneuraminic acid (Neu5Gc) is synthesized from its precursor N-acetylneuraminic acid (Neu5Ac) by cytidine-5'-monophospho-N acetylneuraminic acid hydroxylase (CMAH), which is encoded by the CMAH gene. Most mammals have both Neu5Gc and Neu5Ac, but humans and ferrets have only Neu5Ac because of loss-of-function mutations. Dogs and cats are polymorphic for Neu5Gc and Neu5Ac expression like cats, in which the CMAH gene is responsible for the AB Blood group system. Although the CMAH gene has been characterized in many species, not much is known about it in dogs. In this study, we cloned the dog CMAH cDNA, and performed mRNA expression analysis of this gene in several organs. We also identified single nucleotide polymorphisms (SNPs) in the CMAH gene. Results:We cloned the 1737-bp open reading frame of the dog CMAH gene. This gene consists of at least 14 coding exons and codes for a polypeptide of 578 amino acids and is located on chromosome 35. The amino acid identities of dog CMAH with the corresponding sequences from cat, pig, chimpanzee, mouse, and rat were high (89 to 93%). RT-PCR analysis showed that the dog CMAH cDNA was expressed in various tissues. We identified four exonic SNPs (three synonymous and one non-synonymous), 11 intronic SNPs, and an indel in 11 dog breeds by analyzing the nucleotide sequences of the 14 exons, including the coding region of CMAH. In the genotype of the non-synonymous SNP, c.554 A?>?G (p.Lys185Arg), in a total of 285 dogs of seven different breeds, the allele G was widely distributed, and the allele A was the most frequent in the Shiba dogs. The dogs expressing Neu5Ac did not carry the loss-of-function deletion of CMAH found in humans and ferrets, and it remains unclear whether the point mutations influence the expression of Neu5Ac. Conclusions:We characterized the canine CMAH gene at the molecular level for the first time. The results obtained in this study provide essential information that will help in understanding the molecular roles of the CMAH gene in canine erythrocyte antigens.

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Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis.

Publications

Characterization of a novel N-acetylneuraminic acid lyase favoring industrial N-acetylneuraminic acid synthesis.

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