Purification and properties of the gamma-butyrobetaine-binding protein from an Agrobacterium sp

Abstract

A binding protein for y-butyrobetaine was purified from osmotic shock fluid of an Agrobacterium sp. It was a monomeric protein with an apparent molecular weight of 52,000 or 53,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively. The isoelectric point was 4.3, as determined by isoelectric focusing. Amino acid analysis of the protein showed that Asx and Glx were predominant components and that the protein contained no cysteine. The dissociation constant of this protein for 'y-butyrobetaine was found to be 0.7 ,uM by equilibrium dialysis. Attempts to sequence the amino-terminal end with the Edman method failed, suggesting that this region of the protein is blocked.Agrobacterium sp. was isolated from soil by selective aerobic culture with -y-butyrobetaine (4-trimethylaminobutyrate; see inset of Fig. 5) as the sole source of carbon and nitrogen. The metabolism of -y-butyrobetaine in various bacteria has been reviewed recently (5). This quaternary ammonium base is involved in the biosynthesis of carnitine (3-hydroxy-4-trimethylaminobutyrate) or other related betaines (5). In the Agrobacterium sp., the biosynthetic pathway of -y-butyrobetaine leads to the production of the L-isomer of carnitine. This synthesis occurs in two steps:first, oxidation of y-butyrobetaine, which produces the ac4 unsaturated compound trans-crotonobetaine; and second, addition of a molecule of water to the double bond, which results in the formation of L-carnitine. L-Carnitine can be further metabolized to produce intermediates of the Krebs cycle.In mammals, L-carnitine is a normal constituent of plasma and tissues. It comes both from the diet and from biosynthesis in certain tissues, such as the liver and the kidney (12). The principal function of L-carnitine appears to be that of a carrier of long-chain fatty acids across the inner mitochondrial site of P-oxidation. This important function of Lcarnitine is underscored by skeletal muscle weakness, cardiomyopathy, and the accumulation of lipid droplets in tissues from patients diagnosed as having a carnitine deficiency. Such diseases can be treated by administration of L-carnitine.We believe that the Agrobacterium sp. could be a suitable organism for microbial biosynthesis of L-carnitine. For example, L-carnitine can be obtained from the culture medium of mutants blocked in the catabolism of this molecule. However, the first step of microbiological biosynthesis involves the transport of the substrate into the living cell. Since the lipid bilayer cytoplasmic membrane that forms the limit between the cytoplasm and the external medium is essentially impermeable to most hydrophilic substances, cells have evolved specific processes, catalyzed by proteins, that promote the transport of these substances across membranes. These transport processes, which can be the ratelimiting step of biosynthesis, are tightly coupled to the intracellular metabolism. They are subject to regulation by * Corresponding author. metabolic processes, and in turn, ...

Binding-protein-dependent Sugar Transport by Agrobacterium radiobacter and A. tumefaciens Grown in Continuous Culture

“…The N-terminal sequence of GBP2 posed no problems, but preliminary investigations with GBPl indicated that the N-terminus was blocked; this has also been reported for the periplasmic y-butyrobetaine-binding protein from Agrobacterium sp. (Nobile et al, 1988). However, it subsequently proved possible to generate proteolytic fragments from GBP 1 using Table 1.…”

Synthesis of l-carnitine by microorganisms and isolated enzymes

“…strain HK4 possesses a specific and energy consuming transport system for 7-butyrobetaine that is induced by L-carnitine, crotonobetaine and 7-butyrobetaine [-123]. This transport system depends on a periplasmic binding protein [124][125][126][127].…”