δ¹-Pyrroline-5-carboxylic Acid Formed by Proline Dehydrogenase from theBacillus subtilisssp. natto Expressed inEscherichia colias a Precursor for 2-Acetyl-1-pyrroline

Abstract

Proline dehydrogenase (PRODH) catalyzes the biosynthesis of Delta1-pyrroline-5-carboxylic acid (P5C). The Bacillus subtilis subsp. natto gene for the proline dehydrogenase (BnPRODH) was cloned and expressed in Escherichia coli. Nucleotide sequence analysis of the clone revealed an open-reading frame that encodes 302 amino acid polypeptide with a calculated molecular mass of 34.5 kDa. The deduced amino acid sequence showed sequence similarity to bacterial PRODH and PutA of E. coli. The BnPRODH gene was cloned into pET21b and was expressed at a high level in E. coli BL21(DE3). The expressed protein was purified by using nickel ion affinity column chromatography to homogeneity before characterization. The purified recombinant BnPRODH was used to produce P5C. Model system composed of P5C and methylglyoxal was set up to study the formation of 2-acetyl-1-pyrroline. Our data showed that P5C, derived from the conversion of l-proline by the purified recombinant PRODH, might react directly with methylglyoxal to form 2-AP. P5C/methylglyoxal pathway represents the first report of a biological mechanism by which 2-AP may be synthesized in vitro by PRODH.

Mechanism of 2-acetyl-1-pyrroline biosynthesis in Bassia latifolia Roxb. flowers

“…This also confirms non-enzymatic formation of 2AP as also suggested by other researchers (Chen et al 2008). Methylglyoxal was identified as the carbon source for 2AP (Huang et al 2007(Huang et al , 2008. They synthesised 2AP non-enzymatically at room temperature simply by stirring mixture of P5C (Δ 1 -pyrroline-5-carboxylic acid) and Methylglyoxal for 30 min.…”

“…It was found that the loss of function of enzyme betaine aldehyde dehydrogenase 2 (BADH2) is responsible for the accumulation of γ-aminobutyraldehyde (GABald)/Δ 1 -pyrroline; the immediate precursor of 2AP. The addition of acetyl group via methylglyoxal; a product of sugar degradation takes place in Δ 1 -pyrroline to form 2AP (Huang et al 2007(Huang et al , 2008. The synthesis of 2AP in aromatic rice as well as plants like P. amaryllifolius and V. glabra is constitutive in all aerial parts however, in B. latifolia the synthesis of 2AP takes place only in flowers (fleshy corolla), that too at mature stage (Wongpornchai et al 2003;Lorieux et al 1996;Buttery et al 1983a).…”

Virus Evolution toward Limited Dependence on Nonessential Functions of the Host: the Case of Bacteriophage SPP1

“…SPP1 plating in knockout strains carrying a complementing allele of the relevant gene that is expressed ectopically under an inducible P xyl promoter at the amyE locus are shown labeled as complement. The SPP1 plaque phenotype on the putB found to be necessary for efficient phage infection fall into three classes: (i) genes from the yuk operon (yukE, yukD, yukC, yukB, and yueB) coding for components of the Esat-6-like secretion system (40); (ii) protein synthesis (efp [41]); (iii) intermediary metabolism (ycgM [42,43]). As it was elegantly demonstrated by the groups of M. A. Santos and C. São-José, YueB and other proteins encoded by the yuk operon are required for phage SPP1 irreversible binding to the B. subtilis cell surface and are possibly involved in postadsorption events, such as phage genome transfer to the host cell cytoplasm (13,32,40).…”

“…YcgM is a proline dehydrogenase, the first enzyme of the pathway for oxidation of proline to glutamate (42,43). Its function has been demonstrated in B. subtilis, and the name of the gene has been changed to putB (43).…”

Identification of aroma volatiles and understanding 2-acetyl-1-pyrroline biosynthetic mechanism in aromatic mung bean (Vigna radiata (L.) Wilczek)

“…Similarly, Huang et al (2008) reported higher level of MG in scented rice calli than that of non-scented rice calli. Huang et al (2007) demonstrated that MG reacts with P5C, to form 2AP. In the present study, higher levels of MG might be contributing towards higher levels of 2AP in SMB.…”